Heparin-Binding Epidermal Growth Factor–Like Growth Factor, Collateral Vessel Development, and Angiogenesis in Skeletal Muscle Ischemia
Objective— Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is a potent mitogen for smooth muscle cells and has been implicated in atherosclerosis, tissue regeneration after ischemia, vascular development, and tumor angiogenesis. We examined the hypothesis that HB-EGF participates in angiogenesis and collateral growth in ischemia.
Methods and Results— During 3 weeks after femoral artery ligation, no attenuation occurred in recovery of hindlimb perfusion or distal saphenous artery flow in HB-EGF–null (HB-EGF−/−) versus wild-type mice. Lumen diameters of remodeled collaterals in gracilis muscle were similar by morphometry (87±8 versus 94±6 μm) and angiography, although medial thickening was reduced. Gastrocnemius muscle underwent comparable angiogenesis (41% and 33% increase in capillary-to-muscle fiber ratio). Renal renin mRNA, arterial pressure, and heart rate during anesthesia or conscious unrestrained conditions were similar between groups. These latter findings validate comparisons of perfusion data and also suggest that differences in arterial pressure and/or renin–angiotensin activity are not masking an otherwise inhibitory effect of HB-EGF absence. Four days after ligation, EGF receptor phosphorylation increased in muscle by 104% in wild-type but by only 30% in HB-EGF−/− mice. This argues against compensation by other EGF receptor ligands.
Conclusion— Our results suggest that HB-EGF is not required for arteriogenesis or angiogenesis in hindlimb ischemia.
Angiogenesis and arteriogenesis occur in adults as adaptive responses to increased tissue growth, tissue activity, and ischemic vascular diseases. Angiogenesis involves endothelial cell (EC) proliferation and migration, recruitment of circulating endothelial progenitor cells and leukocytes, and invasion of capillary sprouts into surrounding tissue.1 Arteriogenesis is the expansion or maturation of rare arteriole-to-arteriole anastomoses between adjacent arterial beds, initiated by increased shear stress, into large caliber collateral vessels after critical narrowing or occlusion of a major supply artery.2 Arteriogenesis is accompanied by leukocyte and T-lymphocyte accumulation around the growing collaterals, followed by vascular wall cell proliferation, lumen expansion, and wall thickening.3 Although the signaling pathways initiating arteriogenesis and angiogenesis are presumed distinct, some similarities exist, eg, circulating multipotential cells participate in both processes. Evidence suggests that vascular wall cells, leukocytes, and T cells release growth factors that direct phenotypic changes, migration, apoptosis, and proliferation of ECs, smooth muscle cells (SMCs), and adventitial/periadventitial fibroblasts.4–6 However, the number of growth and differentiation factors that direct arteriogenesis and angiogenesis are extensive and incompletely defined.
Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is an EGF family member that was initially identified as a growth factor secreted from human macrophage–like U-937 cells.7 Soluble HB-EGF is shed from a transmembrane-anchored precursor (pro-HB-EGF) and activates EGF receptor (EGFR)/ErbB1/HER-1, EGFR-4/ErbB4/HER-4, and N-arginine dibasic convertase (NRDc).8–10 Although pro-HB-EGF can inhibit proliferation and stimulate apoptosis in a juxtracrine manner in cell culture,11 evidence suggests that autocrine/paracrine HB-EGF is the main biologically active form in vivo. HB-EGF has potent mitogenic and chemotactic activities for many cell types, including SMCs and fibroblasts.9,12,13
Recent evidence has implicated HB-EGF in angiogenesis. Induction of HB-EGF expression by vascular endothelial growth factor (VEGF) released from ECs led to the suggestion that HB-EGF may participate in recruitment of mesenchymal cells that form the medial and adventitial layers of arteries and veins during embryonic development,14 a process with similarities to arteriogenesis in the adult. Exogenous HB-EGF promoted rabbit corneal angiogenesis and mouse skin neovascularization.15 As well, angiotensin-induced angiogenesis in the rabbit corneal assay was linked to HB-EGF transactivation of EGFR.16 Moreover, evidence suggests that the vascular trophic effect of catecholamines, which is mediated by α1-adrenoceptor-dependent HB-EGF release and activation of EGFR,17 contribute to arteriogenesis and angiogenesis in mouse hindlimb ischemia.18 In addition, angiopoietin, which is induced by VEGF and is important in embryonic recruitment of pericytes and SMCs to developing vessels, stimulated expression and release of HB-EGF by ECs.19 Recently, HB-EGF has been implicated in an autocrine loop involving VEGF induction of HB-EGF that, in turn, augments VEGF expression in tumor angiogenesis.20
These studies, together with the importance of macrophages in arteriogenesis and angiogenesis and as a source of HB-EGF, suggest that HB-EGF may contribute to growth of collaterals and capillaries in ischemic tissue. However, no studies have examined this hypothesis. This possibility is further strengthened, particularly in the case of arteriogenesis, by the observation that increased shear stress induces expression of HB-EGF in ECs.21 As well, tumor necrosis factor-α, a cytokine released by macrophages that has been implicated in arteriogenesis,22 induces expression of many genes in ECs, including HB-EGF.23 Therefore, mice with germ-line deletion of HB-EGF were used to test the hypothesis that HB-EGF contributes to collateral growth and angiogenesis induced by hindlimb ischemia.
Materials and Methods
An expanded Materials and Methods section can be found in the online data supplement available at http://atvb.ahajournals.org.
Unilateral Hindlimb Ligation
The femoral artery of 4- to 5-month-old mice was ligated proximal to the genu artery and distal to the origin of the lateral caudal femoral and superficial epigastric arteries (the latter was also ligated) and resected between the ≈1 mm spaced ligatures. All measurements were made by observers blinded to the genotype of the animals.
Laser Doppler Perfusion Imaging
Scanning velocimetry was performed in an anatomically defined region of the lateral gastrocnemius and plantar foot under isoflurane anesthesia (Figure 1A), as described previously.18 Distal saphenous artery flow velocity was determined in nonscanning mode (Figure 1C).
Length and diameter of the perforating collateral artery deep within the adductor were obtained by angiography three weeks after ligation.
Histomorphometry and Angiogenesis
Lumen diameter and intima media thickness were measured in the mid-zone of the anterior gracilis collateral artery. Angiogenesis was determined in the gastrocnemius muscle.
Arterial Pressure and Heart Rate
Arterial pressure and heart rate were determined on the second and third days after femoral artery ligation under anesthesia and in the conscious unrestrained state.
Renal Renin mRNA
Real-time RT- PCR was performed on total kidney RNA as described previously.24 Values for the left and right kidneys were comparable and thus averaged.
Immunoprecipitation and Western Blotting
EGFR was immunoprecipitated from lysates of the mid-zone of the medial adductor muscle 4 days after ligation. Blots were probed for phosphotyrosine and EGFR.
Data are expressed as means±SEM for “n” (numbers of animals) and were subjected to parametric and nonparametric analysis.
HB-EGF−/− mice do not show impaired recovery of hindlimb perfusion after femoral artery ligation. To test whether HB-EGF is required for ischemia-induced collateral growth and angiogenesis, laser scanning Doppler velocimetry was used to determine hindlimb perfusion in wild-type and HB-EGF−/− mice (Figure 1). Both groups evidenced similar restoration of perfusion 3 weeks after femoral artery ligation (Figure 1A and 1B).
Recovery of flow velocity in the distal saphenous artery also did not differ between groups (Figure 1C and 1D). After femoral ligation, distal saphenous artery flow is dependent on collateral conductance. This was reflected in the loss of flow velocity pulsation after ligation, attributable to dampening caused by the small diameter of the collaterals before they outwardly remodel (Figure 1C). Saphenous artery flow is also dependent on downstream conductance, which is influenced by angiogenesis, and on saphenous artery diameter. The latter is a function of pressure, smooth muscle tone, compliance, and arterial remodeling. To assess remodeling after femoral ligation, we used a stereomicroscope to measure outside diameter of the saphenous artery of 4- to 5-month-old C57BL/6×129Sv mice after acute exposure through a 1-mm incision under isoflurane anesthesia. When normalized to body weight and during maximal dilation with topical 0.5% lidocaine, diameter (in μm/g) decreased from 8.5±0.3 to 7.2±0.4 immediately after ligation and increased to 8.0±0.8 and 9.2±0.5 at 10 and 21 days after ligation (n=6 to 9 per time point). Thus, biphasic remodeling of saphenous artery occurred over the 3-week duration studied herein. This remodeling, together with pressure gradient, upstream and downstream conductance, and smooth muscle tone, determined saphenous artery flow velocity at the time points examined after femoral ligation. It is currently not possible to measure these parameters in the same mouse over time. However, the similar recovery of saphenous artery perfusion in both groups (Figure 1D) provides a confirmation of the similar recovery of plantar perfusion (Figure 1B) because the latter primarily reflects superficial capillary density and flow.
Growth of Collaterals Is Not Altered in HB-EGF−/− Mice
Lumen diameter and medial thickness increased comparably in the anterior gracilis collateral 3 weeks after ligation (Figure 2B), although medial thickening was less in HB-EGF−/− and was not significant (P=0.07; Figure 2C). Likewise, length (reflecting the increase in tortuosity), diameter, and derived volume of the “mid-zone” of the perforating collateral artery increased similarly in both groups (Figure 3). There were no differences between groups in any parameter in the nonligated limb or in the absolute value or percentage increase of parameters in the ligated limb (Figures 2 and 3⇓).
Angiogenesis in Gastrocnemius Is Not Impaired in HB-EGF−/− Mice
Angiogenesis was examined in the lateral head of the gastrocnemius that experiences ischemia immediately after femoral ligation. Capillary density increased similarly in both groups, although the absolute increase in wild-type mice was not significant (Figure 4A). Reduced muscle fiber size from ischemia and/or reduced use can confound the interpretation of changes in capillary density vis-à-vis angiogenesis. Therefore, the ratio of capillary number-to-muscle fiber number and average muscle fiber size were determined (Figure 4B and 4C). Similar increases in capillary-to-fiber ratio and decreases in fiber size were seen in both groups.
By 3 days after ligation, neither group evidenced indications of hindpaw ischemia at rest (eg, cyanosis or edema). Three weeks later, coloration appeared normal and no loss of toenails or necrosis of digits occurred in either group.
Controls for Cardiac Hypertrophic Phenotype of HB-EGF−/− Mice
Deletion of the HB-EGF gene causes, with varying penetrance, abnormalities in embryonic development of cardiac cushions and a modest reduction in alveolar number and increase in interstitial tissue in lung.25 Defective valvulogenesis results in stenosis of the semilunar and atrioventricular valves, leading to cardiac hypertrophy, whereas no phenotype has been attributed to the lung phenotype.25 Cardiac hypertrophy from chronic valvular stenosis can progress to heart failure, depending on degree and duration (ie, animal age) of stenosis. If present in the current study, heart failure could potentially influence arteriogenesis and angiogenesis because of hemodynamic disturbances and activation of the renin–angiotensin system.
To address this issue, relative heart and lung weights were determined because body weight differed between groups (wild type, 40.2±1.5 g, n=13; HB-EGF−/−, 30.1±1.5g, n=13). HB-EGF−/− mice had significantly greater wet weight of heart and lung, whereas lung dry weight was similar between groups (Figure 5A). Water represented 60% of the increase in lung wet weight in HB-EGF−/− mice. This normal tissue water fraction, which accompanied the increase in interstitial tissue that is characteristic of HB-EGF−/−, indicates absence of congestive heart failure and pulmonary edema. This suggests that the cardiac hypertrophy in the ≈4.5-month-old mice studied herein was compensatory. Moreover, regression analysis of data from laser perfusion (Figure 1B), collateral volume (Figure 3D), and capillary angiogenesis (Figure 4A and 4B) showed no correlation with cardiac hypertrophy in HB-EGF−/− mice (Figure 5C and 5D; Figure I, available online at http://atvb.ahajournals.org). In an additional group of mice, mean arterial pressure and heart rate were determined immediately after chronic catheterization of the right femoral artery while mice were under the same isoflurane anesthesia used to obtain the perfusion data in Figure 1. Pressure and heart rate were also measured on the second and third days after catheterization in the conscious unrestrained state in the home cage of the animals. There were no differences in heart rate or arterial pressure between wild-type and HB-EGF−/− mice (Figure II, available online at http://atvb.ahajournals.org). The absence of a difference in pressure under anesthesia indicates that the perfusion data in Figure 1 are not a reflection of a difference in arterial pressure between the groups. Furthermore, the absence of lower arterial pressure and/or tachycardia in HB-EGF−/− mice in the conscious or, in particular, anesthetized state is consistent with the data in Figure 5A (and Figure I), suggesting that cardiac hypertrophy has not progressed to heart failure. This was further confirmed by the absence of an increase in kidney renin mRNA in HB-EGF−/− mice (Figure 5B). Renal renin mRNA correlates closely with renin–angiotensin system activity.26
Collectively, these results suggest that HB-EGF−/− mice have compensated cardiac hypertrophy and, thus, no hypotension or activation of the renin–angiotensin system. The findings do not support the hypothesis that cardiac hypertrophy in HB-EGF−/− mice is producing a hemodynamic or humoral disturbance that is promoting arteriogenesis and angiogenesis and, thus, masking an otherwise inhibitory effect of HB-EGF absence.
Increased Phosphorylation of EGFR in Ligated Limbs Is Attenuated in HB-EGF−/− Mice
Phosphorylation of EGFR more than doubled in the collateral-forming mid-zone of the adductor musculature 4 days after ligation in wild-type mice (Figure 6). Activation of EGFR was consistent with induction of cell proliferation27 expected after collateral remodeling. In contrast, phosphorylation of EGFR increased 70% less in HB-EGF−/−. Total levels of EGFR did not differ between nonligated and ligated legs and were comparable between groups. Similar results were obtained for gastrocnemius muscle. The small residual level of activation in HB-EGF−/− mice, which may represent ligand-independent EGFR transactivation or receptor stimulation by other EGF family members, suggests that compensation has not occurred by these additional mechanisms to obscure an otherwise inhibition of vascular growth in HB-EGF−/− mice.
HB-EGF has functions ranging from mitogenic and chemotactic effects on SMCs and fibroblasts to antiapoptotic activities that help enable cells and tissues to survive hypoxic, oxidative, and nutritional stresses. HB-EGF also contributes to wound healing and post–ischemic tissue regeneration,8 and is upregulated during differentiation of the SMC-like pericyte, an important event in angiogenesis.28 Also, increased shear stress, a key initiating stimulus in arteriogenesis, enhances in vitro expression of HB-EGF by ECs.21 Although evidence from studies of embryonic, adult corneal, and tumor angiogenesis14–16,20 suggests that HB-EGF may contribute to ischemia-induced capillary sprouting and collateral growth, no studies have tested this possibility. Therefore, we examined the hypothesis that HB-EGF contributes to ischemia-induced angiogenesis and/or arteriogenesis. Unexpectedly, absence of HB-EGF did not attenuate lumen expansion or lengthening of collaterals, although wall thickening was lessened modestly. The latter effect may extend from loss of a trophic effect of HB-EGF on collateral SMCs. Angiogenesis was also not reduced. Consistent with these data, recovery of hindlimb perfusion and distal saphenous artery flow did not differ between HB-EGF−/− and wild-type mice. Also, no differences were evident in hindpaw appearance or hindlimb use, which both appeared normal.
Increased phosphorylation of EGFR was evident in hindlimb musculature 4 days after ligation and was sharply reduced in HB-EGF−/− mice. This suggests that HB-EGF–EGFR signaling is enhanced by ligation in vascular wall cells because EGFR is well known to be expressed in ECs and SMCs but not in skeletal muscle cells.29 Activation of EGFR, however, is not required for ischemic arteriogenesis or angiogenesis, based on the other findings reported herein. Moreover, in the absence of HB-EGF, EGFR phosphorylation by other EGF family members or intracellular EGFR transactivation mechanisms does not fully compensate. Taken together, our results indicate that neither HB-EGF nor EGFR activation are required for arteriogenesis or angiogenesis in the ligation model examined, unless the low level of residual phosphorylation in the HB-EGF−/− mice (30% of normal) is sufficient to convey EGFR signaling necessary for vascular growth.
Although HB-EGF–null mice provide the advantage of elimination of HB-EGF signaling, phenotypic differences arising from gene deletion require consideration. HB-EGF−/− mouse embryos have enhanced mesenchymal cell proliferation in cardiac cushions and lung parenchyma, resulting, with variable penetrance, in defective valvulogenesis and enlarged stenotic cardiac valves in the adult.25,30 This is also present in EGFR−/− mice.31 Stenosis of the semilunar and atrioventricular valves causes compensatory cardiac hypertrophy that can progress to heart failure, depending on the severity and duration of the stenosis. Heart failure is accompanied by compensatory activation of humoral systems, including the sympathetic nervous and renin–angiotensin systems, with the latter serving as an early indication of heart failure. Progression of heart failure to congestion is accompanied by pulmonary and peripheral edema, as well as tachycardia and conditions favoring hypotension, especially under anesthesia. Hypotension and activation of the renin–angiotensin and sympathetic nervous systems, the latter of which contributes to arteriogenesis and angiogenesis in the murine hindlimb ligation model,18 could complicate interpretation of measurements of arteriogenesis and angiogenesis in ligation models. However, in the current study, HB-EGF−/− mice had cardiac hypertrophy without evidence of heart failure. Lung wet weight in HB-EGF−/− mice was modestly increased in proportion to normal tissue water expected to accompany the small increase in lung dry weight. This presumably arises from the increase in lung interstitium evident in these mice.25 These data, together with no evidence of peripheral edema, argue against the presence of congestive heart failure.
Absence of edema does not rule out less-severe heart failure. However, HB-EGF−/− mice older than those used in the present study (6 versus 4.5 months) had only a 25% reduction in left ventricular fractional shortening.25 This is less than the mildest level of human heart failure (class I), which is characterized by reduction in ejection fraction of at least 40%. Furthermore, our younger HB-EGF−/− mice had no hypotension or tachycardia, whether examined in the conscious or anesthetized state, and no activation of the renin–angiotensin system. In addition, we found no correlation between the amount of cardiac hypertrophy and measures of angiogenesis, arteriogenesis, or recovery of hindlimb perfusion. Collectively, these findings suggest that HB-EGF−/− mice have compensated cardiac hypertrophy without heart failure. They, thus, argue against the presence of hemodynamic (eg, hypotension and greater hindlimb ischemia) or humoral disturbances (eg, elevated plasma angiotensin and catecholamines) that, by promoting arteriogenesis and angiogenesis, masked our detection of an otherwise inhibitory effect of HB-EGF absence.
Our results suggest that HB-EGF−/− and EGFR signaling are not required for arteriogenesis or angiogenesis in adult ischemic disease, at least for the level of flow reduction examined. However, these findings do not rule out the possibility that other EGFR ligands or mechanisms of intracellular transactivation of EGFR are present or recruited such that the small residual level of EGFR activation in the ligated limb of HB-EGF−/− mice (Figure 6) remains sufficient to compensate for HB-EGF absence. EGF, transforming growth factor-α and amphiregulin induce EGFR/ErbB1 receptor tyrosine phosphorylation and coupling to physiological responses that are similar to HB-EGF.32 However, such compensation seems unlikely because, other than a modest attenuation of collateral wall thickening, no deficits were detected in arteriogenesis and angiogenesis, even though EGFR activation was reduced by >70%. It also remains possible that other ErbB receptors and their ligands are normally involved or compensate in the absence of HB-EGF and are, thus, obscuring a contribution of HB-EGF to angiogenesis or arteriogenesis in the HB-EGF−/− mice. For example, betacellulin activates both EGFR and ErbB4, receptors that also bind HB-EGF.33,34 Other signaling pathways unrelated to the EGF family could also compensate when EGF signaling is compromised. Additional studies are required to examine these possibilities.
This work was supported by National Institutes of Health grants R01-HL062584 (J.E.F.), R01-CA43973 (D.C.L.), and a Holderness Medical Student Fellowship (S.M.M.).We thank Kirk McNaughton for histology and Dr Hyungsuk Kim for conducting the renal renin mRNA assay.
- Received May 17, 2005.
- Accepted June 9, 2005.
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