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From the Molecular Cardiology/Whitaker Cardiovascular Institute, Boston University School of Medicine, Massachusetts.
Correspondence to Dr Kenneth Walsh, PhD, Molecular Cardiology/Whitaker Cardiovascular Institute Boston University School of Medicine, 715 Albany St, W611 Boston, MA 02118. E-mail kxwalsh@bu.edu
Abstract
Objective— Diabetes is a risk factor for the development of cardiovascular diseases associated with impaired angiogenesis or increased endothelial cell apoptosis.
Methods and Results— Here it is shown that angiogenic repair of ischemic hindlimbs was impaired in Leprdb/db mice, a leptin receptor–deficient model of diabetes, compared with wild-type (WT) C57BL/6 mice, as evaluated by laser Doppler flow and capillary density analyses. To identify molecular targets associated with this disease process, hindlimb cDNA expression profiles were created from adductor muscle of Leprdb/db and WT mice before and after hindlimb ischemia using Affymetrix GeneChip Mouse Expression Set microarrays. The expression patterns of numerous angiogenesis-related proteins were altered in Leprdb/db versus WT mice after ischemic injury. These transcripts included neuropilin-1, vascular endothelial growth factor-A, placental growth factor, elastin, and matrix metalloproteinases implicated in blood vessel growth and maintenance of vessel wall integrity.
Conclusion— These data illustrate that impaired ischemia-induced neovascularization in type 2 diabetes is associated with the dysregulation of a complex angiogenesis-regulatory network.
Angiogenic repair of ischemic hindlimbs was impaired in diabetic Leprdb/db and WT mice as evaluated by laser Doppler measurements. cDNA expression profiles were created from adductor muscle of Leprdb/db and WT mice before and after hindlimb ischemia illustrating the dysregulation of a complex angiogenesis-regulatory network in diabetic mice.
Key Words: diabetes ? ischemia ? angiogenesis ? microarrays
Introduction
Diabetes is associated with microvascular rarefaction and reduced collateralization in ischemic tissues.1,2 These circulatory deficits often lead to ischemic injury, impaired wound healing, and the development of peripheral artery disease in diabetic patients. The mouse ischemic hindlimb model is used to study angiogenic repair associated with peripheral artery disease.3 In this model, the distal portion of the saphenous artery and major side-branches are excised, leading to vascular insufficiency. In response to this injury, there is a time-dependent increase in limb perfusion and neovascularization that can be evaluated by laser Doppler blood flow (LDBF) analysis and by assessing capillary density in tissue sections. Development of collateral vessels is a complex process requiring the action of multiple genetic programs.4 Microarray technology provides a method for identifying novel therapeutic and diagnostic target genes in cardiovascular disease, and this technology has been used previously to analyze the expression patterns of 12 000 transcripts involved in collateral vessel formation in C57BL/6 mice.5
Previous studies have found that ischemic hindlimb neovascularization is impaired in models of type 1 diabetes, including streptozatocin-treated rats and mice, nonobese diabetic mice, and alloxan-treated rabbits.6–8 Type 2 diabetes is more prevalent in industrialized regions, and it is characterized by elevations in insulin and peripheral insulin resistance. Type 2 diabetes commonly occurs in patients who are obese and display features of the metabolic syndrome. The Leprdb/db mouse is a model of obesity and type 2 diabetes mellitus. Leprdb/db mice become identifiably obese and show elevations of plasma insulin and of blood glucose because of the spontaneous mutation of the leptin receptor.9
The current study had 2 aims. First, we tested whether a mouse model of obesity and type 2 diabetes (Leprdb/db) displays impaired angiogenesis in hindlimbs that are made ischemic. Second, we performed DNA microarray analyses on Leprdb/db and wild-type (WT) mice before and after ischemic surgery to assess the differences in the expression profiles of angiogenesis-related proteins to identify candidates that may account for the impaired angiogenic response in Leprdb/db mice.
Methods
For the Methods section, please see the online supplement, available at http://atvb.ahajournals.org.
Results
Impaired Ischemia-Induced Vascular Remodeling in Leprdb/db Mice
The mice analyzed in this study were C57BL/6 (WT) and Leprdb/db on the C57BL/6 background. Blood pressure did not differ between the 2 groups on the day of surgery (Table I, available online at http://atvb.ahajournals.org). Significant differences were observed in body weight, plasma glucose, and insulin and leptin levels between WT and Leprdb/db mice. All mice survived after surgical induction of left hindlimb ischemia. Figure 1A shows representative LDBF images of hindlimb blood flow before surgery, immediately after surgery, and at 14 days after surgery in the WT and Leprdb/db mice. Immediately after left femoral artery and vein resection, the ratio of blood flow between the ischemic and nonischemic hindlimbs decreased to 0.21±0.08 in WT and 0.22±0.08 in Leprdb/db mice, indicating that the severity of the induced ischemia was comparable in the 2 experimental groups. In WT mice, hindlimb blood flow perfusion increased to 75% of the nonischemic limb by day 14 (Figure 1B). In contrast, flow recovery in Leprdb/db mice was impaired, and the deficits in flow were statistically significant at 14, 28, and 35 days after surgery.
Figure 1. Impaired angiogenic response in the ischemic hindlimb of Leprdb/db mice compared with WT mice. A, Representative LDBF analyses in WT and Leprdb/db mice before surgery, immediately after surgery, and at 14 days after surgery. A low perfusion signal is indicated by dark blue, whereas a higher perfusion signal is indicated by white to red. B, Quantitative analysis of the ischemic/nonischemic LDBF ratio in WT and Leprdb/db mice before and after surgery and on postoperative days 1, 7, 14, 28, and 35 (n8); #P<0.05; *P<0.01.
To investigate the extent of vascular remodeling at the level of the microcirculation in WT or Leprdb/db mice, quantitative analysis of capillary density in ischemic and contralateral adductor muscle of WT and Leprdb/db mice was determined in histological sections harvested on postoperative day 14. Quantitative analysis of CD31-positive cells revealed that the ischemia-induced increase in capillary density in the ischemic limb relative to the contralateral limb was essentially absent in Leprdb/db mice (Figure 2), indicative of an impaired angiogenic response in these animals. At the 14-day time point after surgery, the ischemic adductor muscle of Leprdb/db mice had significantly fewer CD31-positive cells compared with ischemic muscle from WT mice.
Figure 2. Reduced capillary density in ischemic Leprdb/db mice. Quantitative analysis of capillary density in ischemic (I) and contralateral (C) adductors of WT and Leprdb/db mice on postoperative day 14 (n=8). Capillary density was expressed as the number of capillaries per high-power field (x400; left) and capillaries per muscle fiber (right). *P<0.01.
Transcript Expression Before and After Hindlimb Ischemia in WT and Leprdb/db Mice
Transcript expression profiles in WT and Leprdb/db mice were analyzed before surgery and 1, 7, and 14 days after surgery. RNA was isolated from normal or ischemic adductor muscles and hybridized to Affymetrix GeneChip Mouse Expression Set microarray 2.0 (45 000 cDNAs and ESTs). Transcript level differences between WT and Leprdb/db mice at the same time point, or between different time points for the same mouse strain, were deemed statistically significant if the fold change difference was >2.0 (n=3; P<0.01). This analysis revealed 356 transcripts that were differentially regulated between WT and Leprdb/db mice before surgery. At 1, 7, and 14 days after surgery, 683, 3362, and 3128 transcripts, respectively, were differentially regulated compared with presurgery in WT mice. In Leprdb/db mice, 979, 310, and 1371 transcripts were differentially regulated at days 1, 7, and 14 respectively, compared with presurgery. Of note, many of the genes differentially expressed in Leprdb/db mice were not detected in the data set from WT mice. At the 14-day time point, 66% of genes detected as differentially expressed in Leprdb/db mice were not detected as differentially expressed in WT mice (Figure 3A). Similarly, 74% and 87% of differentially expressed transcripts in Leprdb/db mice were not detected as differentially expressed in WT mice at days 7 and 1 after surgery, respectively. Thus, although fewer genes are differentially expressed in Leprdb/db mice, they do not appear to simply represent a subset of genes regulated in WT. Instead, most of the transcripts regulated in Leprdb/db mice after ischemic surgery represent a different set of genes.
Figure 3. Biological network analysis of differentially expressed transcripts in WT and Leprdb/db mice. A, Venn diagrams representing the sets of transcripts in WT and Leprdb/db mice that are differentially regulated at 1, 7, and 14 days after surgery. Black represents transcripts differentially regulated in WT; white, transcripts differentially regulated in Leprdb/db; gray, transcripts differentially regulated in both strains. B, Network constructed from differentially regulated transcripts comparing microarray data from the hindlimbs of WT mice at baseline and 14 days after surgery. The network score was 27. Shaded genes were identified as differentially expressed by the extent of shading that is indicative of the magnitude of regulation. Red shading indicates upregulation at day 14 relative to baseline, whereas green shading indicates downregulation. Node shapes indicate function; diamond, an enzyme; square, growth factor; triangle, kinase; circle, other. Asterisk indicates transcripts identified multiple times on the microarray; line, physical interactions (eg, formation of complexes); arrow, functional interaction (activation); , functional interaction (inhibition). C, Transcripts differentially regulated in Leprdb/db mice comparing baseline and day 14 after surgery superimposed on the network constructed for WT mice as described in B.
An automated analysis of related transcripts was performed using Ingenuity Pathways Analysis software. For this analysis, a set of 3128 transcripts differentially expressed in WT mice at day 14 compared with baseline (presurgery; fold change >2.0 or <2.0; P<0.01) was used as the starting point for generating biological networks. This time point was chosen because it was the earliest to achieve statistical significance in the LDBF analysis (Figure 1). One of the networks achieved a score of 27 and consisted of 35 transcripts, of which 34 were differentially regulated at 14 days after surgery (Figure 3B). A key providing transcript identity for this network and its fold change is provided in Table 1. These transcripts included the angiogenesis-regulatory factors vascular endothelial growth factor-A (VEGF-A), neuropilin-1, neuropilin-2, placental growth factor, and the putative transcriptional regulators of neuropilin-1, the HHEX homeobox factor.10 In contrast to the identification that a number of proangiogenic transcripts were upregulated, VEGF-B and RORC, a transcriptional regulator of VEGF-A,11 were downregulated. Other differentially expressed transcripts in this network included 3 proteasome subunits, translation regulatory factors (EIF4E, EIF4EBP2, and ETFI), the proteases cathepsin S and lysozyme, and a number of metalloproteinases (matrix metalloproteinase-2 [MMP2], MMP12, MMP16). In addition, the matrix proteins elastin, SPARC, and myelin basic protein (MBP), and 5 protein kinases that phosphorylate MBP, were upregulated in the ischemic hindlimbs of WT mice. Once established, the 1371 transcripts differentially expressed in Leprdb/db mice at day 14 were superimposed on this network (Figure 3C; Table II, available online at http://atvb.ahajournals.org). Only 10 differentially regulated transcripts were observed in Leprdb/db mice compared with 34 in WT mice. Of note, differentially regulated transcripts observed in WT but not in Leprdb/db mice included the angiogenesis growth factors and neuropilin coreceptors elastin and the MMPs.
TABLE 1. Pathways Analysis Network for WT Mice on Day 14 After Surgery Compared With Baseline (P<0.01)
A number of transcripts within the network were also identified as differentially regulated at earlier time points. In WT mice, ELN, NRP1, and EIF4E were detected as differentially regulated at day 1 after surgery, and CLK2, CTSS, EIF4E, EIF4EBP2, HHEX, HSPB8, LYZ, MBP, MMP16, NID, NRP1, NRP2, PAK3, PSMB7, RGS2, RORC, SPARC, tissue factor pathway inhibitor, tissue inhibitor of metalloproteinases-4 (TIMP4), VEGF-A, and VRK1 were detected as differentially regulated at 7 days. In contrast, in Leprdb/db mice, only RGS2 was identified as differentially regulated at 1 day, and MBP and TIMP4 were identified as differentially regulated at 7 days.
The 356 transcripts identified as differentially expressed between WT and Leprdb/db mice before surgery (fold change >2.0 or <2.0; P<0.01) were superimposed on this network (data not shown). No differentially expressed transcripts were observed in the network, demonstrating that the differences observed between WT and Leprdb/db mice reflected a differential response of the tissues to ischemic injury rather than a baseline difference presurgery. Finally, other networks were identified using the Ingenuity software (Please see online supplement, available at http://atvb.ahajournals.org).
Quantitative RT-PCR of Selected Transcripts
The differential expression of 3 angiogenesis-related transcripts identified by the microarray and pathway analyses were examined in greater detail by quantitative RT-PCR (QRT-PCR) using the primer sets listed in supplemental Table III (available online at http://atvb.ahajournals.org). The representative transcripts chosen for this analysis were elastin, the main component of the extracellular matrix of arteries, neuropilin-1, a coreceptor for VEGF receptor 2 (VEGF-R2),12 and VEGF-A, a major angiogenic factor. Consistent with the microarray data, little or no difference in the levels of these transcripts were apparent before surgery in the limbs of WT and Leprdb/db mice (Table 2). QRT-PCR assays showed that these transcripts were upregulated at 1, 7, and 14 days after surgery (elastin and neuropilin-1) or at 7 and 14 days after surgery (VEGF-A) in WT mice. However, elastin, neuropilin-1, or VEGF-A upregulation was not observed in the Leprdb/db mice on days 1, 7, or 14 after surgery, consistent with the findings of the microarray data.
TABLE 2. Comparison of Microarray and QRT-PCR Data*
Elastin and Neuropilin-1 Protein Expression in Ischemic Muscle After Hindlimb Ischemia
Elastin and neuropilin-1, 2 factors involved in arterial morphogenesis or angiogenesis, were also analyzed by Western immunoblot analysis in WT and Leprdb/db mice. Elastin and neuropilin-1 transcripts were identified as differentially expressed at 1, 7, and 14 days in WT but not Leprdb/db mice. Immunoblotting for elastin in samples from muscle tissue of Leprdb/db and WT mice before hindlimb ischemia surgery and on days 1, 7, and 14 after surgery showed an upregulation in WT mice, whereas no upregulation was detected in the ischemic limbs of Leprdb/db mice (Figure 4A). Immunoblotting also revealed an increase in the neuropilin-1 content of the ischemic muscle tissue in WT mice after hindlimb ischemia, in accordance with the microarray and QRT-PCR data. In contrast, the increase in neuropilin-1 protein content after ischemic muscle tissue of Leprdb/db mice was considerably lower (Figure 4B).
Figure 4. Immunoblot analysis of neuropilin-1 and elastin expression in WT and Leprdb/db mice. Western immunoblots with the indicated antibodies were performed on adductor muscle before surgery and at 1, 7, and 14 days after surgery. Representative immunoblots for elastin (A, left panel) and neuropilin-1 (B, left panel) in WT and Leprdb/db mice are shown. Quantitative analysis of relative changes in elastin (A, right panel) and neuropilin-1 (B, right panel). Elastin and neuropilin-1 signals were normalized to the signal for tubulin and expressed as percent relative to control (before surgery).
Discussion
Diabetes and obesity promote microvascular rarefaction and diminish collateral vessel development in the heart and peripheral tissues.1,2 The Leprdb/db mouse is a model of type 2 diabetes that results from a leptin receptor deficiency. Here we show that the angiogenic repair of ischemic hindlimbs is impaired in Leprdb/db mice compared with C57BL/6 (WT) mice, as evaluated by laser Doppler flow and capillary density analyses. These data are consistent with observations of diminished skin wound healing in these animals.13–15
Here it is also shown that the impairment is associated with alterations in a gene regulatory network involved in the physiological revascularization process. Microarray analysis revealed alterations in the expression patterns of numerous extracellular matrix protein–related and angiogenic growth factor–related transcripts between WT and Leprdb/db mice after hindlimb ischemia. In particular, factors underexpressed in the ischemic limbs of Leprdb/db mice at 14 days after ischemia include the angiogenic factors VEGF-A (confirmed by QRT-PCR), placental growth factor, neuropilin-1 (confirmed by QRT-PCR and Western immunoblot), neuropilin-2, and elastin (confirmed by QRT-PCR and Western immunoblot). None of the transcripts identified in this network were found to be differentially expressed between these 2 mouse lines at baseline (data not shown). These data indicate that the Leprdb/db mouse fails to correctly activate a multifaceted angiogenic program in response to ischemic injury.
Analysis of the microarray data reveals that fewer transcripts are differentially expressed in the ischemic limbs of Leprdb/db mice than WT mice at days 7 and 14 after surgery. Comparison of the data sets indicate that most of the genes identified as differentially regulated in Leprdb/db mice do not represent a subset of the genes regulated in WT mice. Instead, the majority of transcripts represent a distinct set of genes that are differentially regulated in WT and Leprdb/db mice. In this regard, leptin has been recognized as a proangiogenic molecule that displays synergistic effects with fibroblast growth factor-2 and VEGF in promoting blood vessel growth.16,17 Although leptin deficiency may be directly responsible for some of the changes in the angiogenic program of Leprdb/db mice, the diabetes and obesity that develop as a consequence of leptin deficiency is also likely to have a profound impact on the angiogenesis-regulatory network in this model.
Our experiments showed that transcripts for MMP2, MMP12, or MMP16 were upregulated in WT mice after hindlimb ischemia, but this response was suppressed in Leprdb/db mice. These data also revealed the downregulation of TIMP4, a negative regulator of MMP2, in WT and Leprdb/db mice. Metalloproteinases hydrolyze components of the extracellular matrix, creating the cellular environments required during development or morphogenesis.18 MMPs are important regulators of angiogenesis by virtue of their abilities to allow endothelial cell migration through the degradation of matrix and to activate latent cytokines and growth factors. In this regard, MMP2 cleaves SPARC, a protein implicated in tumor progression and angiogenesis,19,20 and it has been proposed that SPARC cleavage can yield proangiogenic peptides.21 Like MMP2, SPARC transcripts are upregulated in the ischemic limbs of WT but not Leprdb/db mice. Less is known about the proangiogenic activities of MMP12 and MMP16, but MMP12 has been implicated in macrophage migration,22 and MMP16 promotes the cleavage and activation of MMP2.23
The microarray, QRT-PCR, and Western immunoblot analyses revealed upregulation of elastin in WT but not Leprdb/db mice. Elastin is the main component of the extracellular matrix of arteries, and it performs a regulatory function during arterial morphogenesis and development by controlling proliferation of smooth muscle and stabilizing the arterial structure.24 In diabetic animals, the elasticity of this protein is reduced as a result of increased glycosylation, leading to the accumulation and reorganization of smooth muscle cells.25 In addition, the involvement of elastin derived peptides in angiogenesis has been reported.26 These proangiogenic fragments of elastin can be produced by the proteolytic actions of MMPs, including MMP2. Elastin is also cleaved by cathepsin S and MMP12,27,28 which are upregulated in the ischemic hindlimbs of WT but not Leprdb/db mice.
In addition to the dysregulation of extracellular matrix, impaired regulation of a number of angiogenic growth factors and growth factor receptors were differentially regulated in the ischemic limbs of WT and Leprdb/db mice. Microarray data showed that VEGF-A, placental growth factor, neuropilin-1, and neuropilin-2 were upregulated in WT mice after surgery, but this regulation was impaired in Leprdb/db mice. VEGF-A is a key hypoxia-inducible angiogenic factor that induces the proliferation, migration, and survival of endothelial cells,29 and also promotes the expression of proteases implicated in pericellular matrix degradation in angiogenesis.30 Activation of VEGF-R1 by either VEGF-A or placental growth factor induces different gene expression profiles and promotes the phosphorylation of distinct tyrosine residues in the tyrosine kinase domain of VEGF-R1, and the combined administration of these factors enhances VEGF driven angiogenesis.31 Neuropilin-1 and neuropilin-2 are cell-surface glycoproteins that participate in the regulation of angiogenesis.32 Neuropilin-1 functions as a coreceptor for VEGF-R2 that enhances the activity of VEGF-A in ischemic tissues.12
An unexpected finding of this study is the observation that MBP and a series of protein kinases that phosphorylate this protein are upregulated in the ischemic limbs of WT but not Leprdb/db mice. MBP is also cleaved by MMP2.33 MBP functions in maintaining the structural integrity of the myelin sheath, and it is subjected to a wide array of post-translational modifications.34 Thus, the impaired induction of MBP and its regulatory network in Leprdb/db mice may contribute to the peripheral neuropathy that is associated with peripheral vascular disease.35
There has been considerable interest in "therapeutic angiogenesis" because many patients are not amenable to traditional forms of revascularization.36 However, despite a large number of positive preclinical studies, the results of controlled therapeutic angiogenesis trials have been largely negative. One reason that may account for the lack of clinical success is that preclinical studies are almost exclusively performed on healthy, young animals that exhibit robust angiogenic responses, whereas the patient population experiences a high incidence of obesity and insulin-resistant diabetes. Therefore, we used a mouse model of type 2 diabetes and obesity to study revascularization after ischemic injury. Leprdb/db mice displayed functional and anatomic deficits in hindlimb revascularization that was associated with the dysregulation of a complex angiogenesis-regulatory network. These data suggest that a single angiogenic agent may not be sufficient to effectively stimulate angiogenesis in subjects with insulin-resistant diabetes and that the control of obesity and its associated diseases may improve the success of proangiogenic therapies.
Acknowledgments
This work was supported by National Heart, Lung, and Blood Institute grant N01-HV-28178 from the National Institutes of Health and National Institutes of Health grants HL66957, AR40197, AG15052, and AG17241 to K.W. We acknowledge the technical assistance of A. Bialik.
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