Immunohistochemical study of the growth factors, aFGF, bFGF, PDGF-AB, VEGF-A and its receptor (Flk-1) during arteriogenesis
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- Wu, S., Wu, X., Zhu, W. et al. Mol Cell Biochem (2010) 343: 223. doi:10.1007/s11010-010-0517-3
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Growth factors are viewed as main arteriogenic stimulators for collateral vessel growth. However, the information about their native expression and distribution in collateral vessels is still limited. This study was designed to profile expression of acidic and basic FGF, platelet-derived growth factor (PDGF-AB) and vascular endothelial growth factor (VEGF-A) and its receptor, fetal liver kinase-1 (Flk-1) during arteriogenesis by confocal immunofluorescence in both dog ameroid constrictor model and rabbit arteriovenous shunt model of arteriogenesis. We found that: (1) in normal arteries (NA) in dog heart, aFGF, bFGF, and PDGF-AB all were mainly expressed in endothelial cells (EC) and media smooth muscle cells (SMC), but the expression of aFGF was very weak, with those of the other two being moderate; (2) in collateral arteries (CAs), aFGF, bFGF, and PDGF-AB all were significantly upregulated (P < 0.05); they were present in all the layers of the vascular wall and were 2.1, 1.7, and 1.9 times higher than that in NA, respectively; and (3) in NA in rabbit hind limb, VEGF-A was absent, Flk-1 was only weakly present in endothelial cells, but in one week CAs VEGF-A and Flk-1 were significantly increased in both shunt and ligation sides; this was more evident in the shunt-side CAs, 2.3, and 2 times higher than that in the ligation side, respectively. In conclusion, our data demonstrate for the first time that growth factors, aFGF, bFGF, and PDGF-AB are significantly upregulated in collateral vessels in dog heart, and enhanced VEGF-A and its receptor, Flk-1, are associated with rapid and lasting increased shear stress. These findings suggest that endogenous production of growth factors could be an important factor promoting collateral vessel growth.
Induction of arteriogenic reactions to enhance collateral vessel development has been suggested as a promising approach for the treatment of severe vascular occlusive disease , and this concept has been termed ‘‘therapeutic arteriogenesis.’’ Therapeutic arteriogenesis has motivated scientists to search for proangiogenic/arteriogenic factors to stimulate collateral vessel growth. Currently, FGFs, VEGF, and PDGF are viewed as main arteriogenic factors for collateral vessel growth. In the early 1990s, a number of studies, including in animals and small open trials for the treatment of patients with coronary arterial diseases (CAD) and periphery arterial diseases (PAD), demonstrated that these proteins could stimulate collateral vessel growth and augment blood supply to ischemic tissue [2–5]. However, recently, the data from two double-blinded, randomized, and placebo-controlled clinical trials failed to provide evidence for efficiency in treatment of ischemic patients . Furthermore, the role of VEGF in promoting collateral vessel growth became controversial even in animal experiments [7, 8]. These findings lead to the speculation that VEGF is a stimulator for angiogenesis, but not for arteriogenesis.
The failure of growth factor application in the treatment of ischemic patients and the controversial results of VEGF in animal experiments imply that the underlying mechanism for collateral vessel growth including growth factors is still only partially understood. In fact, although numerous animal experiments show favorable results of collateral augmentation by using a variety of growth factors, and their expression being elevated in patients in response to ischemia , few studies have characterized the native expression profile of growth factors in natural growing collateral vessels.
In this study, expression of growth factors, aFGF, bFGF, PDGF, and VEGF was detected by immunoconfocal microscopy with specific antibodies against these proteins in collateral vessels in dog heart induced by implantation of an ameriod constrictor around the left circumflex artery and in rabbit hind limb induced by femoral ligation or by an arteriovenous shunt created unilaterally between the distal stumps of one of the bilaterally occluded femoral arteries with the accompanying vein. Our data showed that aFGF, bFGF and PDGF-AB were significantly upregulated in growing collateral vessels in dog heart and enhanced VEGF-A and its receptor, Flk-1 were associated with rapid and lasting increased shear stress in rabbit hind limb of arteriogenesis.
Materials and methods
Ameroid constrictor animal model
The protocol for preparation of this animal model has been previously described . In brief, six adult mongrel dogs weighing 18–20 kg were anesthetized with pentobarbital 138 mM/kg bodyweight. Under artificial respiration, the thorax was opened, the pericardium incised, and an ameroid constrictor was implanted around the circumflex branch of the left coronary artery (LCx). The thorax was closed, and the animals were allowed to recover using analgesics and antibiotics.
The experiments were carried out following the guide for the care and use of laboratory animals published by the National Institute of Health (NIH publication No. 85-23, revised in 1996).
Rabbit ischemic hind limb model
Since we did not find good anti-VEGF and Flk-1 antibodies that worked with dog material, we studied the rabbit arteriogenesis model. In brief, six adult New Zealand white rabbits were used in this study. The animals were anesthetized with an i.m. injection of midazolam (1 mg/kg) and xylazine (5 mg/kg), and acute bilateral femoral artery ligation was performed with two knots. Immediately after occlusion, an arteriovenous (AV) shunt was created unilaterally side-to-side, between the distal femoral artery stump and the accompanying femoral vein. The simply ligated side was used as control. Then, the skin was closed with sterile surgical clips. The animals were allowed to recover completely, and housed with free access to water and food. We did not observe any gangrene or gross impairment of hind limb function after femoral artery occlusion and arteriovenous fistula creation. All the animals received antibiotic-(Benzylpenicillin) and analgesic treatment (Buprenorphin).
For the ameroid constrictor model, at three and six weeks post-surgery, corresponding to the phase of early and active growth of collateral vessels, respectively , the animals were re-anesthetized, the thorax was opened, and the heart was removed. All midzone samples of the collaterals were removed in addition to normal coronary arteries as control tissue. A total of 40 vessels were investigated. For the rabbit hind limb ischemic model, at day seven post-surgery, the animals were re-anesthetized; and the collateral vessel tissues were removed. All the samples were immediately frozen in liquid nitrogen, embedded in tissue processing medium (O.C.T), and stored at −80°C till further use.
Primary and secondary antibodies used in this study
For comparison with growing collateral vessels, size-relative coronary arteries from normal dog hearts, and the femoral artery and its branches from normal rabbits without either ligation or shunt, were used as normal control.
In all staining procedures, incubation with PBS instead of the first antibody was used as negative control to exclude nonspecific binding of the secondary detection system.
The quantification of immunofluorescence intensity was performed with a Leica TCS SP confocal microscope, using the quantitation software from Leica as described previously . In brief, one channel with format 512 and appropriate filters was used. A full range of gray values from black to peak white (0–255-pixel intensity level) were set during the whole process of measurements. The intensity of fluorescence was expressed as arbitrary units AU/μm2.
All data are presented as mean ± SEM. The t-test was used to examine the difference between normal vessels and growing vessels.
Expression of aFGF, bFGF and PDGF-AB in normal and collateral vessels in the dog heart
Quantitative analysis of immunofluorescence density (AU/μm2) of aFGF, bFGF, PDGF-AB, VEGF (A) and Flk-1 in normal (NV), control (only ligation) (CV) and AV-shunt (AV-S) vessels
24.31 ± 8.14
50.08 ± 8.30*
45.23 ± 4.11
76.82 ± 7.13*
39.15 ± 4.27
74.39 ± 7.93*
8.02 ± 9.90
24.04 ± 10.05*
48.08 ± 10.05
1.01 ± 3.74
19.56 ± 10.05*
44.98 ± 10.05
Expression of VEGF (A) and Flk-1in normal and collateral vessels in rabbit hind limb
The negative control with PBS incubation instead of the first antibody showed no nonspecific binding of the secondary detection system. The data were not shown.
The main findings of this study are (1) the growth factors—aFGF, bFGF, and PDGF-AB—were significantly upregulated during arteriogenesis induced by the implantation of an ameroid constrictor around the left circumflex artery in dog heart; and (2) expression of VEGF-A and its receptor Flk-1 were upregulated during arteriogenesis induced by ligation of the femoral artery, and this was significantly augmented by elevated fluid shear stress.
Acidic FGF, bFGF, and PDGF-AB in normal arterial vessels in dog heart
Expression of aFGF, bFGF in normal arterial vessels in the heart was described in several species, including human, rat, and avian [13–15]. However, these data are controversial to some extent. Hughes et al. has reported that, in human coronary arteries, bFGF is localized in medial smooth muscle cells and adventitial blood vessels, absent in the luminal endothelium, whereas aFGF expression was weak in endothelium and media, but was predominant in adventitial fibroblasts . Spirito et al.  found that in rat coronary arteries, immunostaining for both aFGF and bFGF was intensely positive in the smooth muscle cells at early stages of development, but became faint or negative with increasing cell differentiation. The expression profiles of aFGF and bFGF in this study are partly in agreement with above mentioned reports by the observation that both factors were present in endothelium and media, but not in adventitia. We speculate that the difference of localization between these two growth factors is probably due to species differences, as previously reported for species difference in expression of connexins 37 in dog heart . In addition, tissue sampling should be taken into account as a factor since only small arteries were collected for examination, which contain no blood vessels in adventitia . As for PDGF, it is known that normal vessels generally express low or undetectable levels of PDGF ; however, expression of PDGF in coronary arteries is rarely reported. Although Van Den Akker et al. observed that in avian-maturating coronary vasculature, endothelial cells expressed PDGF-B , and currently, there is no information about PDGF-AB protein in the coronary vasculature. Here we demonstrated that PDGF-AB is expressed in endothelium and media in dog normal coronary arteries.
The constitutive expression of aFGF, bFGF, and PDGF by quiescent endothelial cells and contractile smooth muscle cells in normal arteries is not consistent with their function of mitogenic activity, probably suggesting a functional involvement in the maintenance of normal vascular homeostasis. On the other hand in normal arterial vessels extracellular matrix proteins such as collagen IV and laminin are abundant, which suppress cell proliferation and dedifferentiation , and therefore, the presence of these growth factors in normal vessels may act as a potential reservoir as vascular remodeling occurs.
Expression of aFGF, bFGF and PDGF-AB during arteriogenesis in dog heart
Experimental studies in rat, pig, and dog using exogenous bFGF and PDGF-AB [21–23] indicate a considerable role of these growth factors in promoting collateral vessels growth. However, there is little information about their native presence and distribution in collateral vessels during arteriogenesis. Here, we added to the knowledge that an increased expression of aFGF, bFGF, and PDGF-AB was present in all the layers of the vascular wall in growing collateral vessels in dog heart.
It has been known that the chronic arterial occlusion of a main artery by an ameroid constrictor leads to a rapid growth of collateral vessels that can, upon demand, expand their diameter by a factor of 10 on average and increase their flow capacity by a factor of 25 . Several important events are considered to contribute to this process, including cell proliferation and apoptosis, endothelial activation, phenotype changes of smooth muscle cells, and extracellular proteolysis [10, 11]. This study suggests that increased aFGF, bFGF, and PDGF-AB in collateral vessels could also be an important contributor for rapid collateral vessel growth in dog heart since they are powerful mitogens that stimulate cell migration, proliferation, and differentiation of various cell types [18, 24, 25]. This notion is further confirmed by the observation that the increased expression of bFGF is accompanied by high rate of cell proliferation in serial sections.
In contrast to the expression profile of aFGF and bFGF presented in this study, Deindl et al. reported that the mRNA levels of FGF-1 and -2 remained low and constant in a rabbit hind limb model of arteriogenesis . Furthermore, increased fluid shear stress failed to upregulate most growth factors (PDGF, FGF-2, and FGF-4) and their receptors in shunt-side growing collateral vessels as well . The mechanism for the different expression patterns of these growth factors in collateral vessels between dog heart and rabbit hind limb is not clear. It might be due to species difference or regional and model difference. However, it remains to be determined, yet.
VEGF-A and its receptor, KDR/Flk-1, in a rabbit hind limb model of arteriogenesis
In order to unravel the puzzle that VEGF fails to promote collateral vessel growth and improve collateral blood flow, we investigated the expression of VEGF and its receptor, KDR/Flk-1, in our arteriovenous shunt model of rabbit hind limb created unilaterally between the distal stumps of one of the bilaterally occluded femoral arteries with the accompanying vein. We observed that VEGF and Flk-1 were simultaneously upregulated in the endothelial and smooth muscle cells in growing collateral vessels after one week of ligation. More significantly, upregulation of VEGF and Flk-1 was observed in shunt-side growing collateral vessels. This is in line with our previous article suggesting that mRNA of Flk-1 (VEGFR-2) was upregulated after 7 days of increased fluid shear stress . Based on recent findings that VEGF participates in regulating endothelial proliferation and migration, matrix degradation, endothelial focal adhesion assembly, smooth muscle cell migration and mitogenic effects of MCP-1 on vascular smooth muscle cells [28–31], we propose that VEGF-A may stimulate collateral vessel growth in an indirect way because VEGF is not a direct mitogen for vascular smooth muscle cells.
The observation of higher levels of expression of VEGF-A and its receptor, Flk-1, in shunt-side collateral vessels suggests that the flow shear stress is a critical player regulating production of VEGF-A and Flk-1, whereas the observation of simultaneous upregulation in expression of VEGF-A and its receptor, Flk-1, implies that VEGF-A and Flk-1 function synergistically. Because VEGF receptor1 (Flt-1) acts as a decoy receptor and only Flk-1 is actively involved in regulating the angiogenic process , and modulates functional signaling pathways in response to VEGF, by functioning as the kinase for many substrates and recruiting signaling molecules , therefore on one hand, low level of expression of VEGF and Flk-1 in ligation control-side collateral vessels may be one of explanations why these vessels were less developed than those in shunt-side. On the other hand, limited expression of Flk-1 in ligation side vessels may help us to understanding why administration of VEGF fails to improve collateral circulation because limited number of Flk-1 was probably completely bound to endogenous VEGF during arteriogenesis, with no unbound receptor available for exogenous VEGF. In order to clarify this issue, further experiments including the use of inhibitors for VEGF and Flk-1 with more time-points are needed.
In conclusion, our data for the first time reveal the expression profile of growth factors, aFGF, bFGF, and PDGF-AB during arteriogenesis in dog ameroid constrictor model, and VEGF and its receptor, Flk-1, in collateral vessels in rabbit hind limb model of arteriogenesis. These data contribute new information with regard to the mechanism for arteriogenesis by showing that arteriogenesis is naturally associated with an increased expression of these arteriogenic factors. The differential expressions of aFGF, bFGF, and PDGF during arteriogenesis in dog heart and rabbit hind limb suggest thet differences in the tissue or organ may be an important factor responsible for their production. This finding may serve as a springboard for further mechanistic studies.
This study was partly supported by the NSFC of Chinese government (Nos. 30771134 and 30971532), and by the (non-profit) Kuehl Foundation of Germany.