Catecholamine stimulation of α1-adrenoceptors exerts growth factor-like activity, mediated by generation of reactive oxygen species, on arterial smooth muscle cells and adventitial fibroblasts and contributes to hypertrophy and hyperplasia in models of vascular injury and disease. Adrenergic trophic activity also contributes to flow-mediated positive arterial remodeling by augmenting proliferation and leukocyte accumulation. To further examine this concept, we studied whether catecholamines contribute to collateral growth and angiogenesis in hindlimb insufficiency. Support for this hypothesis includes the above-mentioned studies, evidence that ischemia augments norepinephrine release from sympathetic nerves, and proposed involvement of reactive oxygen species in angiogenesis and collateral growth. Mice deficient in catecholamine synthesis [by gene deletion of dopamine β-hydroxylase (DBH−/−)] were studied. At 3 wk after femoral artery ligation, increases in adductor muscle perfusion were similar in DBH−/− and wild-type mice, whereas recovery of plantar perfusion and calf microsphere flow were attenuated, although not significantly. Preexisting collaterals in adductor of wild-type mice showed increases in lumen diameter (60%) and medial and adventitial thickness (57 and 119%, P < 0.05 here and below). Lumen diameter increased similarly in DBH−/− mice (52%); however, increases in medial and adventitial thicknesses were reduced (30 and 65%). Leukocyte accumulation in the adventitia/periadventitia of collaterals was 39% less in DBH−/− mice. Increased density of α-smooth muscle actin-positive vessels in wild-type adductor (45%) was inhibited in DBH−/− mice (2%). Although both groups experienced similar atrophy in the gastrocnemius (∼22%), the increase in capillary-to-muscle fiber ratio in wild-type mice (21%) was inhibited in DBH−/− mice (7%). These data suggest that catecholamines may contribute to collateral growth and angiogenesis in tissue ischemia.
- dopamine β-hydroxylase
- laser-Doppler imaging
the prevalence of occlusive vascular disease of the coronary, cerebral, and peripheral limb circulations underscores the importance of understanding how adaptive remodeling of the vasculature in ischemia is regulated. Ischemic remodeling consists of sprouting angiogenesis and positive remodeling (i.e., “maturation”) of preexisting collaterals into conduit arteries. In addition, evidence suggests that new collaterals may form and undergo outward remodeling (15, 58, 90). Although collateral development, also termed arteriogenesis, has greater potential for restoring blood flow to severely ischemic beds than does angiogenesis alone (43, 77), less is known about the mechanisms that direct arteriogenesis.
Flow-induced shear stress is an important biomechanical force in the initiation of arteriogenesis (47, 68, 94). In most tissues, a small number of preexisting collaterals make interconnections between distalmost arterioles of adjacent vascular beds. The density of these preexisting collaterals varies among and within species. Shear stress increases within collaterals when the lumen of the conduit artery supplying one bed becomes narrowed and pressure below it is reduced (41, 75). In vitro studies have shown that increased fluid shear stress on endothelial cells (ECs) causes phosphorylation of proteins at focal adhesions and intercellular junctions that are postulated to serve as mechanoreceptors (78). Subsequently, transcription is induced in ECs for adhesion molecule and chemokine genes, many of which possess shear stress response elements (63, 81). Leukocytes and T lymphocytes adhere, diapedese, and accumulate in the perivascular region of maturing collaterals, where they secrete proteases that modify the perivascular extracellular matrix (ECM) to permit outward remodeling (11). Paracrine growth factors released from perivascular cells, vascular wall cells, and binding sites in the restructuring ECM are thought to direct phenotypic changes, proliferation, apoptosis, and migration of ECs, vascular smooth muscle cells (VSMCs), and adventitial fibroblasts. These events promote luminal expansion and wall hypertrophy of the remodeling collateral, which return shear and circumferential wall stresses toward normal.
In addition to biomechanical forces and paracrine growth factors, certain G protein-coupled receptor agonists that stimulate VSMC contraction also exert trophic actions that contribute to pathological growth. In particular, although the trophic actions of angiotensin are much more firmly established, recent evidence indicates that norepinephrine directly induces proliferation, hypertrophy, and migration of rat aorta VSMCs and adventitial fibroblasts studied in cell culture (24, 31, 102, 108). Also, in aorta organ culture, norepinephrine-mediated proliferation and protein synthesis in VSMCs were strongly augmented after balloon injury and mediated by α1A-adrenoceptors (ARs) (107). Similar effects occurred in vivo; perivascular administration of norepinephrine in the carotid artery wall increased neointimal growth and lumen narrowing after balloon injury (30). Moreover, local (30) and systemic (91) blockade of α1A-ARs inhibited hypertrophy and restenosis of the carotid artery, demonstrating a contribution of endogenous catecholamines to vascular wall growth after injury. Constriction of the aorta and carotid artery in the rat and mouse is mediated by a different AR subtype, the α1D-AR (21). Inhibition of this subtype and other α1-, α2-, and β-AR subtypes that are also expressed in media and adventitia had no effect (30, 107), consistent with in vitro studies (102, 107). α1-AR-mediated trophic activity in VSMCs is signaled by NAD(P)H oxidase-dependent generation of reactive oxygen species (ROS), shedding of heparin-binding epidermal growth factor-like growth factor, transactivation of epidermal growth factor receptors, and subsequent stimulation of p42/44 mitogen-activated protein kinases (7, 105).
Studies using mutant mice have confirmed that adrenergic trophic activity is strongly augmented in arteries undergoing growth and remodeling. Injury-induced hypertrophic remodeling of the carotid artery was sharply attenuated in mice made deficient in catecholamines by gene deletion of dopamine β-hydroxylase (DBH−/−) (106). This was associated with inhibition of proliferation and leukocyte accumulation. Use of different mutant mice lacking individual α1-AR subtypes indicated that α1B-ARs mediated the trophic effect in mice, in contrast to α1A-ARs in the above-mentioned studies in rats (106). Similarly, hypertrophic negative remodeling of the mouse carotid artery induced by low shear stress and, importantly for the present study, adaptive positive remodeling induced by high shear stress were strongly inhibited in DBH−/− and α1B-AR−/− mice, but not in mice deficient in the α1D-AR, which is also expressed in this vessel (29). As with wall injury (106), this inhibition was associated with reduction in proliferation and leukocyte density around the remodeling carotid artery (29). Additional studies by others have confirmed that α1B-ARs mediate direct catecholamine-induced arterial hypertrophy and remodeling in mice (96). Whether a similar trophic action of catecholamines contributes to arteriogenesis and angiogenesis in ischemic tissue has not been examined.
These studies suggest that endogenous catecholamine stimulation of α1-ARs contributes to artery growth and remodeling in pathological and adaptive physiological settings. To test the generality of this hypothesis, in the present study, we used mice deficient in catecholamine synthesis to examine whether catecholamines contribute to adaptive remodeling in hindlimb insufficiency. Support for this hypothesis derives not only from the above studies, but also from evidence that injury and ischemia augment norepinephrine release from nerves (9, 12) and that generation of ROS, which is augmented by catecholamines (7, 105), may contribute to angiogenesis and arteriogenesis (36, 48, 95).
MATERIALS AND METHODS
Unilateral hindlimb ischemia.
Six-month-old DBH−/− mice and control (wild-type) littermates were generated on the C57BL/6 and 129Sv background and crossed for >50 generations, as described elsewhere (93). Approximately equal numbers of male and female mice were studied in all groups. Procedures were approved by the University of North Carolina Institutional Animal Care and Use Committee. Animals were anesthetized with ketamine (100 mg/kg im) and xylazine (15 mg/kg im). Hair was removed from the hindquarters with a depilating cream, with care taken to avoid erythema. Body temperature was maintained at 37.0 ± 0.5°C. The femoral artery was exposed aseptically through a 2-mm incision and isolated from the femoral vein and nerve, with care taken to avoid damage to vessels or nerve. The femoral artery was ligated with 7-0 suture just distal to the bifurcation of the anterior epigastric and lateral caudal femoral arteries (LCFA). The wound was irrigated with sterile saline, the incision was closed, and antibiotic (cefazolin, 50 mg/kg im) and antimicotic (furazolidione, topical) were administered.
Laser-Doppler perfusion imaging.
Noninvasive measurements of superficial hindlimb perfusion were obtained before ligation, immediately after ligation, and 1, 3, 7, 14, and 21 days after ligation using a scanning laser-Doppler perfusion imager (model LDI2-IR, Moor Instruments, Wilmington, DE) that was modified for high resolution and depth of penetration (2 mm) with an 830-nm-wavelength infrared 2.5-mW laser diode, 100-μm beam diameter, and 15-kHz bandwidth. Animals were anesthetized with 1.125% isoflurane supplemented with 2:3 oxygen-air, and rectal temperature was closely maintained at 37.0 ± 0.5°C.
Doppler perfusion of the ventral adductor thigh region and plantar hindpaw was obtained within anatomically defined regions of interest (ROIs; High Res, version 5.0, Moor Instruments). The boundaries of the adductor ROIs were drawn to obtain perfusion within the collateral-forming region between the LCFA and saphenous artery (SA). This includes the preexisting superficial collaterals within the anterior and posterior gracilis muscles (see Fig. 3, A and B). The adductor ROI in the nonligated limb was constructed as follows (magenta outlines in Fig. 1) : the lateral boundary was a curve drawn just outside the medial margin of the SA, extending from the SA's proximal appearance (i.e., just beyond the bifurcation of the SA and popliteal arteries from the femoral artery) to its distal end in the calf. The rostral boundary was a line extended at a right angle from the proximal appearance of the SA toward the midline of the animal. The caudal boundary was a line extended at a right angle from the distal end of the lateral boundary to the intersection of the posterior margin of the adductor thigh at its zenith of curvature. The medial boundary was the line connecting this intersection with the rostral boundary. The adductor ROI in the ligated limb was constructed as described above, with the following exceptions: The medial boundary was a line drawn just outside the medial margin of the LCFA that is readily detected by increased flow in the adductor collaterals after ligation. The rostral boundary was drawn as described above so as to intersect the medial boundary. The origin of the caudal boundary was drawn as described above so as to connect to the medial boundary at its intersection with the posterior margin of the thigh. Construction of ROIs was aided by “toggling” between 6- and 16-hue pseudocolor images to enhance identification of the LCFA anatomy. The suture for skin closure interfered with laser detection in that region during acquisition of images immediately “after ligation,” necessitating exclusion of this region in the ROI. This area was identified as the anomalous blue (i.e., low-flow) patch overlying the proximal end of the SA (Fig. 1, right leg after ligation; location was confirmed by comparing the pseudocolor Doppler image with the “photo image,” which shows the actual skin surface that was scanned). Removal of suture just before scanning 24 h later eliminated the need for this ROI adjustment during analysis of subsequent images.
The ROI for the plantar foot consisted of the hindpaw margins (magenta outlines in Fig. 2). All ROIs were drawn by an investigator blind to animal genotype. The average velocity in a ROI was normalized to the area of the ROI, because ROI construction differed for the same animal on different imaging days as a result of unavoidable variation in animal positioning during scanning. Data are reported as ligated-to-nonligated perfusion ratios.
Distal hindlimb microsphere flow.
At 3 wk after ligation, the infrarenal abdominal aorta was cannulated with a pulled-out PE-50 catheter. Animals were perfused at 100 mmHg with phosphate-buffered solution (PBS, pH 7.4) containing 10 μmol/l sodium nitroprusside and 10 U/ml heparin. Microspheres (15 μm diameter; catalog no. F8842, Molecular Probes, Eugene, OR) were vortexed and sonicated, and 150,000 were infused at a constant rate over a 1-min interval into the perfusion line with an infusion pump. After 3 min of additional perfusion, hindlimbs were perfusion fixed with 4% paraformaldehyde in 100 mmol/l sodium phosphate (PFA, pH 7.4) for 15 min. The calf muscle proper was excised, postfixed in 3% PFA-2% glutaraldehyde in 100 mmol/l sodium phosphate (NaP) at 4°C for 30 min with shaking, and rinsed in 100 mmol/l NaP. Tissues were cryoprotected in 30% sucrose in 100 mmol/l NaP overnight at 4°C with shaking and frozen in optimal cutting temperature compound. The total number of microspheres in eight serial 60-μm-thick sections was determined with Texas red fluorescence microscopy, and total volume of the tissue sections was determined by image analysis (ImageJ, version 1.30, NIH). Data are reported as ligated-to-nonligated volume density ratios.
In a separate set of experiments, animals were transcardially perfused at 100 mmHg with PBS containing 10 μmol/l sodium nitroprusside and 10 U/ml heparin 3 wk after femoral artery ligation. PBS was followed by 4% PFA for 20 min. Ligated and nonligated hindlimbs were postfixed in 4% PFA for 48 h with shaking, including a solution change at 24 h. The medial adductor below the femur was excised, and the 5-mm-wide centermost section, which contains the “midzone” of the main hindlimb collateral circulation, was blocked. A section of the gastrocnemius/soleus muscle was also removed en bloc, beginning at the Achilles tendon and extending 5 mm rostrally. Samples were rinsed in water, placed in 70% ethanol for 48 h with shaking and a change of solution at 24 h, and embedded in paraffin.
Sections (5 μm thick) of the adductor sample were stained with modified cyano-Masson elastin. The two preexisting collaterals on the medial surface of the anterior and posterior gracilis muscles (Fig. 3, A and B) were selected by an observer blinded to wild-type vs. null genotype. Sections were digitized at ×40 magnification. Areas were determined as follows (ImageJ): lumen area = (lumen circumference)2/4π, intima-media area = area between the lumen and the external elastic laminus (EEL), circumference = length of the EEL, and adventitial area = area of the dense collagen-containing layer between the EEL and loose perivascular connective tissue. Thickness of the intima-media was calculated as follows: [(EEL circumference ÷ 2π) − (lumen circumference ÷ 2π)]; thickness of the adventitia was calculated similarly using circumferences of the EEL and the outer edge of the dense adventitia, respectively. Thicknesses were calculated, because, in the absence of hypertrophy, wall areas change secondary to geometric changes in vessel circumference. Thus increased area may not equate with hypertrophy. Averages of the above parameters for each collateral were obtained from analysis of two sections separated by 250 μm.
Density of VSMCs and α-smooth muscle actin-positive vessels.
Density of cells in the media of preexisting collaterals in the ligated and nonligated leg was determined by counting nuclei (hematoxylin and eosin). Nuclei that protruded into the lumen were not counted, and counts were normalized to medial area. Nuclear density was determined from two sections separated by 250 μm for each of the two collaterals per animal and averaged for each animal. The analyzed sections were serially adjacent to those used for the morphometric measurements described above. The observer was blinded to animal genotype.
Formation of new α-smooth muscle actin (α-SMA)-positive vessels in the center of the adductor thigh was measured as an increase in the density of 7- to 50-μm-diameter α-SMA-positive vessels (57). Venules of this diameter are generally α-SMA negative and were thus largely excluded. Preexisting collaterals were distinguished from these new vessels by the presence of a venule and somatic nerve that accompany them (79) (Fig. 3C); this was also true for the large collateral (“perforating artery”) located deep in the adductor thigh region that connects the profundus and popliteal arteries. Formation of de novo collaterals is proposed to arise from occasional interbed capillaries between two parallel arterial trees (e.g., the LCFA-to-SA and the profundus-to-popliteal arterial circulations that supply the mouse hindlimb adductor region) that are preexisting (101) or are formed during transient angiogenesis and then acquire a VSMC wall permitting outward remodeling (3, 14, 15, 55, 64, 69, 90). VSMCs were detected using mouse anti-human α-SMA antibody (1:100; Dako, Carpinteria, CA), biotinylated anti-mouse antibody (1:200; Vectastain ABC, Vector Laboratories, Burlingame, CA), and diaminobenzidine (DAB React, Sigma, St. Louis, MO). Sections were lightly counterstained with 0.1% light-green SF yellowish. All α-SMA-positive vessels were counted in a section of the entire ventral adductor thigh musculature at ×40 magnification and normalized to total section area. Density was averaged for two sections 250 μm apart.
Leukocytes in the adventitia/periadventitia of sections serially adjacent to the sections of preexisting collaterals analyzed above for morphometry were detected with rat anti-mouse CD45 antibody (leukocyte common antigen, Ly-5; 1:200; BD Biosciences, Pharmingen, Boston, MA), biotinylated mouse anti-rat secondary antibody (1:100; Vectastain ABC), and diaminobenzidine. Sections were lightly counterstained with Mayer's hematoxylin solution. Cells having a blue nucleus surrounded by a brown reaction product on their surface were counted by an observer blinded to the identity of the randomly arranged slides. The outer periadventitial boundary for leukocyte counting was defined as a region 3.3 times larger than the perimeter of the EEL that circumscribed the collateral in parallel with the EEL. Average leukocyte density was determined from two adjacent 5-μm-thick sections.
Capillary density and atrophy in gastrocnemius muscle.
The plasma membrane of capillary ECs in tissue sections was labeled with biotinylated Griffonia simplicifolia isolectin-1-B4 (GSL-1-B4, 1:100; Vector) and then with Alexa Fluor 488 conjugate of streptavidin (1:50; Molecular Probes). Capillaries were identified as GSL-1-B4-positive vessels with diameters <7 μm, counted within a delineated area of muscle fascicles (ImageJ), and reported as density per square micrometer and as capillary-to-muscle fiber ratio. For muscle atrophy, average muscle fiber size was determined for the fascicles within the delineated area. Boundaries of gastrocnemius muscle fibers and fascicles were outlined by autofluorescence during confocal imaging of skeletal muscle capillaries. In digitized images, muscle fiber area was determined using image analysis software (ImageJ). Values were obtained for capillary density and fiber atrophy from two ×20 fields of view (∼434 × 330 μm) within two sections each of the medial and lateral heads of the gastrocnemius of the ligated and nonligated leg and averaged.
Arterial pressure and heart rate.
Wild-type and DBH−/− mice (n = 6 each) were subjected to right femoral artery ligation as described above. After 1 or 3 days, three mice of each genotype were anesthetized (1.125% isoflurane supplemented with 2:3 oxygen-air) and aseptically implanted with a catheter in the left carotid artery. Catheters were constructed from pulled-out PE-10 tips welded to PE-50 tubing and were exteriorized at the nape of the neck. After implantation, the catheter was connected to a pressure transducer for measurement of mean arterial pressure and heart rate under anesthesia, with rectal temperature maintained at 37.0 ± 0.5°C. Continuous infusion of heparinized saline (10 U/ml) at 2 μl/min with an intraflow device (Sorenson Research, Salt Lake City, UT) maintained catheter patency. Arterial pressure and heart rate were monitored for 20 min, and average values were obtained over a 5-min interval at the end of the recording period.
Renal renin mRNA.
After arterial pressure and heart rate were determined, the animals were euthanized by thoracotomy and exsanguination. The right and left kidneys were rapidly removed and placed into RNAlater (Ambion, Austin, TX). Real-time RT-PCR was performed with an Applied Biosystems 7700 Sequence Detection System (Foster City, CA) on total mRNA from kidneys with primers for renin and β-actin, as previously described (49). Values determined for the left and right kidneys were comparable and, thus, were averaged; β-actin served as a control for RNA extraction. As an internal control, RT-PCR was conducted using primers at 1× and 2× concentrations and yielded nearly identical results.
Values are means ± SE. No differences were detected between male and female mice; thus results were combined. Statistical significance (P < 0.05) was determined by paired t-tests of within-animal comparisons and unpaired t-test for comparisons across groups. Parametric analysis was conducted on all data except capillary density and capillary number-to-fiber ratio, which were subjected to nonparametric analysis.
Remodeling of preexisting collaterals is reduced in mice deficient in catecholamine synthesis.
To test the hypothesis that endogenous catecholamines contribute to arteriogenesis, hindlimb perfusion and morphometry of superficial preexisting collaterals were compared in wild-type and DBH−/− mice after femoral artery ligation. Doppler imaging showed that perfusion in the ventral adductor region containing the superficial preexisting collaterals in the gracilis muscles increased in both groups to a peak at 7 days and remained elevated thereafter (Fig. 1). Collateral growth was also evident between the superficial epigastric and genu circulations and between the genu and distal saphenous beds (Fig. 1). We did not study these regions, because detection of collateral growth in these regions is more variable and, thus, more difficult to quantify histologically. The increase in adductor perfusion (Fig. 1) reflects positive remodeling of the superficial adductor collaterals, as well as reductions in resistance upstream and downstream from them (see below). To our knowledge, the high-resolution methodology used here provides the first noninvasive direct imaging of collateral perfusion and its progression over time.
Of minor note, although absolute perfusion through the collaterals in the adductor ROI of the right leg increased as expected immediately after ligation (Fig. 1), absolute perfusion in the adductor ROI of the nonligated leg (and hindpaw, see below) also increased (absolute data not shown) because of the acute elevation in systemic vascular resistance and arterial pressure caused by ligation. This, along with the effect of ligation to simultaneously reduce saphenous artery-dependent capillary flow in the right adductor, accounts for the lack of increase in adductor perfusion ratio immediately after ligation in the data shown in Fig. 1 (point A, bottom right). However, 1 day later, arterial pressure returned to normal (pressure measurements are given below), and thus the adductor perfusion ratio increased as expected. As is customary, ratio analysis of Doppler perfusion scans was used to normalize for variation, among mice and for the same mouse over time, in rectal temperature, arterial pressure, skin thickness and pigment, and differences in vascular anatomy.
Preliminary longitudinal studies of perfusion in the calf, dorsal hindpaw, and plantar hindpaw after ligation indicated that the greatest consistency in perfusion was obtained from the plantar hindpaw. This is also the region imaged by most other investigators. After femoral artery ligation, hindpaw perfusion recovered similarly in both groups to ∼55–60% at 3 wk after ligation, although it tended to be lower in DBH−/− mice (Fig. 2, top and middle). In agreement, values for collateral-dependent flow to the distal hindlimb as indicated by microsphere measurements at 3 wk were similar to those obtained by perfusion imaging of the hindpaw, including the tendency for flow to be lower (24% decrease, P = 0.063) in DBH−/− mice (Fig. 2, bottom).
Histomorphometry was conducted 3 wk after femoral artery ligation for the superficial collaterals in the anterior and posterior gracilis muscles. These collaterals had outwardly remodeled into corkscrew-shaped vessels characteristic of mature collaterals (Fig. 3, A and B). In contrast to arterioles, preexisting collaterals in mouse gracilis muscles are accompanied by a venule and a somatic nerve (Fig. 3C) (79), which aid in their identification. Because the extent of outward remodeling of both gracilis collaterals was similar, data were averaged (Fig. 4). In wild-type mice, lumen diameter and medial and adventitial thickness increased by 60, 57, and 119%, respectively (Fig. 4). In contrast, although lumen diameter increased similarly (52%) in DBH−/− mice, medial and adventitial thickening were attenuated by ∼50%.
Ligation caused preexisting collaterals to undergo luminal expansion and medial wall thickening, which resulted in greater medial cross-sectional areas: 400 ± 29 vs. 1,013 ± 112 μm2 in wild-type mice (P < 0.001) and 405 ± 36 vs. 790 ± 118 μm2 in DBH−/− mice (P < 0.01). The medial areas of the remodeled collaterals did not differ between wild-type and DBH−/− mice (P = 0.986). The number of nuclei (i.e., cells) in the media approximately doubled 3 wk after femoral artery ligation (Fig. 5, top). When normalized to medial cross-sectional area, nuclear density was reduced in the wild-type, but not the DBH−/−, mice (Fig. 5, bottom).
Overall, these data indicate that preexisting collaterals in DBH−/− mice showed a deficit in wall thickening that normally accompanies luminal expansion during maturation. Comparison of cell (nuclear) density suggests that this is presumably due to inhibition of VSMC hypertrophy and/or reduced expansion of the ECM. Less collateral luminal expansion and recovery of plantar perfusion and microsphere flow after ligation were also seen in DBH−/− mice, although these reductions were not statistically significant.
Leukocyte accumulation around maturing collaterals is reduced in DBH−/− mice.
After ligation, leukocytes are recruited to the periadventitial region of collaterals, where they are proposed to secrete paracrine factors that contribute to remodeling (50, 51, 109). In the present study, although CD45-positive cells were rarely found attached to the endothelium or within the media of the gracilis collaterals 3 wk after ligation, leukocytes were present in the adventitia and periadventitial regions in the ligated leg (Fig. 6, top). Leukocyte accumulation was markedly reduced in mice deficient in catecholamine synthesis (Fig. 6, bottom).
Formation of new α-SMA-positive vessels in the adductor is inhibited in DBH−/− mice.
In models of tissue ischemia, formation of new vessels capable of providing collateral cross-connections is detected as an increase in the density of α-SMA-positive vessels in collateral-forming regions (57, 58, 82, 85). In the present study, wild-type mice showed a 45% increase in these vessels in the centermost region of the ventral adductor thigh posterior to the femur (Fig. 7). This increase was strongly inhibited in DBH−/− mice.
Angiogenesis in the gastrocnemius is reduced in DBH−/− mice.
Angiogenesis was examined in the caudal gastrocnemius, which experiences substantial ischemia immediately after femoral artery ligation (78, 79). The increase in capillary density in the ligated leg of wild-type mice (71%) was less (42%) in DBH−/− mice (Fig. 8). Muscle atrophy from ischemia and/or reduced use can confound the interpretation of changes in capillary density, as opposed to angiogenesis. Therefore, the ratio of capillary number to muscle fiber number and average muscle fiber size were determined (Fig. 8). The 21% increase in capillary-to-muscle fiber ratio in wild-type mice was significantly less (7%) in the DBH−/− group. The amount of atrophy, as indicated by average muscle fiber size, was similar (∼22%) for both groups.
By 3 days after ligation, neither group showed tissue ischemia at rest (e.g., cyanosis or edema) or loss of hindpaw digits or toenails. After 3 wk, coloration and size of toenails and digits appeared normal and were not different between groups.
Arterial pressure, heart rate, and renin-angiotensin system activity.
Recent measurements in mice instrumented for 24-h telemetry over many days showed that arterial pressure is ∼15% lower in DBH−/− than in wild-type mice (87). Therefore, we measured arterial pressure and heart rate under the same anesthesia used during the perfusion-imaging studies. No hypotension or bradycardia was evident in DBH−/− mice, and values did not differ from wild-type controls (Fig. 9). Thus the perfusion-imaging data in Figs. 1 and 2 were not confounded by differences in arterial pressure. During administration of microspheres (Fig. 2) and perfusion fixation for the data in Figs. 3–8, arterial pressure was controlled at 100 mmHg, which approximated the anesthetized pressures of both groups (Fig. 9). Kidney renin mRNA levels in DBH−/− and wild-type mice were also similar (Fig. 9), indicating absence of sufficient hypotension in the conscious state to activate the renin-angiotensin system. Renal renin mRNA correlates closely with renin-angiotensin system activity (53).
Stimulation of α1-ARs induces proliferation, hypertrophy, and migration of VSMCs and adventitial fibroblasts; furthermore, wall injury strongly increases the sensitivity of these cells to adrenergic-mediated growth, leading to enhanced wall thickening and intimal lesion growth (6, 7, 17, 22, 29–31, 65, 91, 96, 102, 104–106, 108). Using DBH−/− mice and mice deficient in α1B-ARs, we recently found that adrenergic trophic activity also contributes to outward remodeling of the carotid artery induced by injury (106) and, in particular, after a chronic increase in shear stress (29). Both of these effects were associated with impaired proliferation of VSMCs and adventitial fibroblasts and reduced accumulation of leukocytes in the vessel wall. These studies indicate that catecholamine trophic activity becomes augmented during arterial wall remodeling in models of pathological and physiological restructuring. The present findings are in agreement. Increases in medial and adventitial thickness of preexisting collaterals in the adductor after femoral artery ligation were inhibited by ∼50% in DBH−/− mice. Leukocyte accumulation in the adventitia/periadventitia and cell hypertrophy and/or matrix content in the media were similarly reduced. Inhibition was also evident in the growth of capillaries in the gastrocnemius muscle and new α-SMA-positive vessels in the collateral-forming region of the adductor thigh muscles. Consistent with these deficits, recovery of plantar perfusion and microsphere flow to the hindleg were less in mice deficient in catecholamine synthesis, although these reductions were not statistically significant. These findings extend the experimental conditions in which catecholamines have been shown to augment vascular growth to include arteriogenesis and angiogenesis in hindlimb insufficiency. In addition, this study provides the first noninvasive imaging of collateral perfusion and its progression over time using new high-resolution laser-Doppler methodology.
Despite reductions in angiogenesis in the gastrocnemius and formation of new α-SMA-positive vessels in the adductor muscles of DBH−/− mice, increase in adductor perfusion and recovery of flow to the distal hindlimb were similar in both groups. There are several possible explanations for this apparent discrepancy. First, we used a “mild” model of impaired hindlimb perfusion, where the femoral artery branches proximal to the saphenous-popliteal bifurcation were left intact. Anatomic measurements were made in the maximally dilated bed, whereas Doppler measurements were obtained at the prevailing level of vascular tone, because it is not possible to combine maximal dilation with noninvasive perfusion measurements in the mouse hindlimb. It is possible that more extensive ligation and ischemic inhibition of basal vascular tone would yield better agreement between anatomic vascular dimensions and blood flow. Indeed, microsphere flow, which was determined under maximal dilation, showed a larger deficit in DBH−/− mice than Doppler perfusion. Because distal hindlimb flow after ligation is much more dependent on increases in conductance of collaterals than downstream capillaries, reduced angiogenesis in DBH−/− mice would be expected to have minimal effect. The absence of inhibition of luminal expansion of preexisting collaterals in mice deficient in catecholamines is consistent with the absence of inhibition of flow restoration. Instead, wall thickening was sharply reduced, which agrees with our previous studies showing that catecholamines stimulate proliferation, smooth muscle cell hypertrophy, and collagen elaboration in remodeling arteries. Finally, the increase in the density of small (7- to 50-μm-diameter) α-SMA-positive vessels was determined from counts throughout the entire musculature of the medial adductor thigh. Because penetration of infrared imaging was restricted to within 2 mm of the skin surface, the contribution of flow in these small vessels to the adductor perfusion signal is predictably minimal compared with the much larger preexisting superficial collaterals connecting the LCFA to the SA. It is likely that more severe ligation and a longer time course would permit new collaterals to achieve larger diameters, with a resultant greater influence on measures of adductor and hindlimb flow.
Although use of DBH−/− mice provides the specificity of gene deletion for inhibition of norepinephrine and epinephrine actions, studies employing animals with null mutations are not free of complications of interpretation. Arterial pressure is an important consideration, because it is a determinant of flow and circumferential wall stress, and wall stress directly influences wall thickness. Although arterial pressure was the same in anesthetized DBH−/− and wild-type mice, in agreement with others (19), arterial pressure in conscious DBH−/− mice was 15% lower than in wild-type controls (87). Reduced arterial pressure would favor lower wall stress in adductor collaterals after femoral ligation when flow-induced dilation of the upstream LCFA and deep iliac arteries exposes collaterals to a larger fraction of the systemic arterial pressure. This could contribute to the reduced wall thickening in preexisting collaterals of DBH−/− mice (Fig. 4). However, lower arterial pressure would also cause greater autoregulatory dilation of resistance vessels downstream from adductor collaterals and flow-mediated dilation of feed arteries upstream from the collaterals. This would mitigate any lower pressure and flow in the collaterals of DBH−/− mice. Lower arterial pressure would also favor greater ischemia and, thus, angiogenesis in the lower hindleg of DBH−/− mice. However, the opposite was observed. Moreover, if hindlimb ischemia were different in the two groups, the amount of atrophy in gastrocnemius muscle should have been different, rather than the almost identical values we obtained (Fig. 8, bottom right). In addition, the absence of an increase in renal renin levels in DBH−/− mice suggests that reduction in their conscious arterial pressure is minimal. In fact, if plasma angiotensin were elevated in DBH−/− mice, this would favor the opposite of what we observed after ligation, i.e., greater angiogenesis (28, 33, 72) and thickening of the collateral wall (35, 71). In addition, DBH−/− and wild-type mice had almost identical preexisting collateral lumen diameters, wall thicknesses, medial nuclear densities, and densities of α-SMA-positive vessels and capillaries in the nonligated limb (Figs. 4, 5, 7, and 8). Differences in one or more of these parameters would be expected in animals with a significant chronic reduction in arterial pressure. In addition, hematocrit is identical in both groups (45 ± 1, n = 13 and 11; unpublished observations). Thus these results do not support the hypothesis that disturbances in hemodynamics in the DBH−/− group account for our findings. Rather, the results are consistent with our previous studies demonstrating that the trophic effects of catecholamines become particularly strong when arteries are induced to grow and remodel by injury or altered shear stress.
Nuclear number increased and area-adjusted density declined in media of wild-type mice (Fig. 5). This indicates that, after 21 days of maturation, ECM had accumulated and/or VSMCs that had previously proliferated underwent hypertrophy. These responses are expected from the increase in circumferential wall tension during collateral outward remodeling and also from a potential increase in collateral pressure caused by flow-mediated dilation of upstream arteries. Nuclear density did not decline in DBH−/− mice, because nuclear number increased similarly in DBH−/− and wild-type mice (Fig. 5, top), but medial thickness (Fig. 4, bottom left) and area increased less. This suggests that, in the absence of catecholamines, less VSMC hypertrophy and/or matrix accumulation occurs during collateral maturation. These results agree with previous findings that norepinephrine induces VSMC hypertrophy in uninjured aorta studied in organ culture (107) and causes an increase in collagen accumulation in balloon-injured carotid in vivo (30).
Leukocyte density around remodeled collaterals was less in DBH−/− mice. Similar results were found in flow-mediated outward remodeling of the carotid artery (29). Previous studies have shown that leukocyte recruitment around collaterals and in capillary beds of ischemic tissue after artery ligation is an important contributor to arteriogenesis (11, 40) and angiogenesis (5, 66). Reductions in circulating leukocytes and expression of endothelial adhesion receptors inhibit both processes (44). Leukocytes may release paracrine factors that participate in remodeling of collaterals, because they do not incorporate into the vessel wall per se (50, 51, 109). Stimulation of α1-ARs on murine bone marrow cells increased lymphopoiesis and decreased myelopoiesis (60, 61). Although additional studies are required to determine the mechanism underlying our leukocyte findings, numbers of circulating leukocytes and development of T and B cells are normal in DBH−/− mice (1). There is also no evidence of intrinsic developmental or functional immune defects in DBH−/− mice, although T cell-mediated immunity to infection is reduced (1).
Catecholamines may augment leukocyte accumulation and/or influence factors released by them. β-ARs are expressed by murine T and B cells (34), and α2-ARs are expressed by macrophages (83). All three α1-ARs are expressed by human monocytes (89). Their expression can be altered in vitro (70); i.e., dexamethasone and terbutaline increased α1B- and α1D-AR mRNA and protein (70), whereas IL-1β and TNF-α decreased α1A-AR, had no effect on α1B-AR, and increased α1D-AR mRNAs (39). However, catecholamines inhibit monocyte production of cytokines and chemokines important in arteriogenesis (TNF-α, IL-6, β-IFN-γ, IL-3, granulocyte-macrophage colony-stimulating factor, and macrophage inflammatory protein-1) (8, 38, 59). The absence of such inhibition in DBH−/− mice would favor an increase in arteriogenesis and angiogenesis, rather than the decrease that we observed. Collectively, these data do not support the concept that our findings result from loss of a stimulatory effect of catecholamines on leukocyte number or release of cytokines and chemokines.
It is possible that the reduced accumulation of leukocytes and wall thickening of preexisting collaterals and inhibition of growth of new α-SMA-positive vessels in DBH−/− mice reflect the absence of ROS-generating effects of catecholamine stimulation of VSMCs and adventitial fibroblasts (7, 105). Increased ROS production by catecholamines may augment expression of chemokines, cytokines, and adhesion molecules in collaterals beyond levels already induced by increased shear stress and the accompanying ROS production (10, 18, 73). Inhibition of ROS generation reportedly attenuates collateral development (36).
In the absence of DBH, catecholamine synthesis terminates in dopamine production. Although dopamine levels have not been measured in arteries of DBH−/− mice, dopamine is only slightly elevated in the heart (93). Dopamine inhibits proliferation and migration of VSMCs; however, this requires nonphysiological concentrations (≥1 μmol/l) and has only been reported in cell culture (103). Dopamine also inhibits tumor angiogenesis (92) and vascular endothelial growth factor (VEGF)-induced angiogenesis, although these effects were obtained at high, pharmacological concentrations (4). Importantly, mutant mice lacking α1B-ARs, but not mice lacking α1D-ARs, showed the same deficit in outward remodeling of the carotid artery as that seen in the DBH−/− mice in two different models (29, 106). These data indicate that the direct vascular trophic effects of norepinephrine in mice are mediated by α1B-ARs and not by dopamine receptors. Taken together, these data do not support the hypothesis that the present results arise from elevated dopamine levels in DBH−/− mice.
Notwithstanding the above-mentioned considerations, one cannot exclude the possibility of an unknown phenotypic effect when using genetically altered mice. Although our previous findings (see the introduction) suggest that α1A- and/or α1B-ARs mediate the effects in the present findings, the type of vascular remodeling examined here differed from that described in previous studies. Thus additional studies are required to identify the subtype(s) responsible for the arteriogenic and angiogenic contributions of catecholamines. This will require genetic methods for targeted, conditional, local knockdown of α- and β-AR subtypes that are not yet available.
Angiogenesis in the gastrocnemius of the ligated leg was reduced in DBH−/− mice. This could reflect inhibition of leukocyte accumulation in the gastrocnemius similar to that surrounding preexisting collaterals in the gracilis muscle, because bone marrow-derived leukocytes, including endothelial progenitor cells and stromal cells, contribute to ischemic angiogenesis. Catecholamines may also augment capillary formation by promoting release of angiogenic factors by ischemic skeletal muscle cells. Catecholamines stimulate angiogenesis in rat brown adipose tissue by a β3-AR-mediated increase in VEGF-120 release from adipocytes (2). Also, stimulation of α1-ARs on cultured neonatal rat cardiac nonmyocytes releases TGF-β and atrial natriuretic peptide into the conditioned medium, which, when presented to cardiomyocytes, upregulates VEGF expression (98). In addition, catecholamines may also augment angiogenesis through direct stimulation of ECs. ECs express α2-ARs in vivo, and some vessels, such as human umbilical vein, express α1B- and α1D-ARs (39). Angiotensin stimulation of endothelial AT1 receptors, which, similar to α1-ARs, are coupled to Gq/11 signaling pathways, induces angiogenesis in hindlimb ischemia (28, 72). Reduced oxygen within the physiological range augments α1B-AR expression by VSMCs in vitro and in vivo through stimulation of a hypoxia-inducible factor-1α promoter element in the α1B-AR gene (25, 26). Because the α1B-AR mediates growth of VSMCs and adventitial fibroblasts in the mouse (29, 106), increased expression in these cells and ECs in ischemic tissue could contribute to the effects reported here for angiogenesis and arteriogenesis. Interestingly, there is recent evidence that the sympathetic cotransmitter neuropeptide Y also promotes angiogenesis (56).
Reduced capillary density in DBH−/− mice may also reflect more “pruning”/revision or a defect in capillary maturation leading to regression after angiogenesis. Capillaries were identified as GSL-1-B4-positive structures with lumens <7 μm in diameter that were located just outside the basement membrane of muscle fibers. Other studies have used this lectin to determine capillary density in angiogenesis (37, 54, 86). Although GSL-1-B4 can stain activated tissue macrophages (88), we would expect their number to have declined by 21 days to a density much lower than muscle capillary density, because there was no evidence of resting ischemia at this time. Consistent with this interpretation, capillary densities before and 21 days after ligation (Fig. 8, top right) agree with values obtained using CD31 and other methods (20, 27).
The increase in density of 7- to 50-μm-diameter vessels possessing α-SMA-positive cells (presumably VSMCs or pericytes) in the adductor midzone was reduced in DBH−/− mice. Although not without controversy (41, 64), the concept of de novo formation of collaterals derives from several observations. Bridging collaterals can form around ameroid constrictors implanted on large arteries (76). Capillaries are capable of remodeling into arterioles and arteries in tumor vascularization (14, 15, 90) and during local VEGF administration (84). In addition, new α-SMA-positive vessels have been described in the collateral-forming regions of ischemic myocardium (57, 80, 82, 99, 100) and hindlimb adductor muscle (58, 85). De novo collaterals are proposed to arise from preexisting capillary cross-connections at the distalmost margins of adjacent beds (64, 101) or during transient angiogenesis in this same region between the oxygenated and ischemic beds (14, 15, 55, 90). Subsequent elimination or pruning of venous connections and acquisition of a VSMC media is thought to permit flow-mediated outward remodeling into a mature collateral (13). In contrast, preexisting collaterals may be remnants of the embryonic arteriolar network that incompletely regressed during development (74). The cellular mechanisms that underlie formation of such “neocollaterals” are not known. Increased shear stress in preexisting capillary interconnections may induce mechanisms similar to those in preexisting collaterals (64). It is well accepted that hypoxia-inducible factor-1α-dependent VEGF signaling is important in angiogenesis (32, 97). However, the occurrence of and the stimulus for transient angiogenesis in the collateral region between beds are controversial issues, with evidence for and against the involvement of ischemia and VEGF (16, 23, 42, 82). Our findings implicating catecholamines in angiogenesis and leukocyte accumulation and wall thickening of preexisting collaterals suggest that catecholamines could also influence the transient angiogenesis and/or shear stress signaling mechanisms that may underlie formation of new collaterals.
The present findings cannot be extrapolated to suggest that adrenergic trophic mechanisms influence vascular growth during normal tissue maturation. DBH−/− mice experience normal tissue growth. Absolute values for Doppler perfusion of the adductor and plantar regions were virtually identical in wild-type and DBH−/− mice before ligation (data not shown). Similarly, no differences were found between wild-type and DBH−/− mice for the following measurements in the nonligated limb: lumen diameter, medial and adventitial thickness (Fig. 4), density of cells in the media (Fig. 5), α-SMA-positive microvessels (Fig. 7) and capillaries, capillary-to-fiber ratio, and muscle fiber size in the nonligated limb (Fig. 8). This is consistent with comparisons of carotid artery and mesenteric resistance artery morphometry in adult DBH−/− and α1B-AR−/− mice (29, 96, 106). Thus these mice, and also α1A-AR−/− and α1D-AR−/− mice, show no differences in adult vascular anatomy or body weight from their wild-type controls (Refs. 29 and 106 and references therein). In addition, although mice lacking catecholamines die during embryonic development (embryonic days 11.5–14.5), death is due to the absence of β-AR-mediated cardiac effects (67). These data suggest that normal growth of the vasculature through adulthood does not require catecholamine trophic actions. In contrast, adaptive and pathological growth in the adult artery appears to involve significant α1-AR-mediated trophic actions on VSMCs and adventitial fibroblasts (Refs. 7, 29, 30, 91, 96, and 105–108 and references therein), including collateral and capillary growth, as shown in the present study. This distinction likely reflects differences in the processes directing normal vascular growth during embryonic and postnatal maturation vs. those directing vascular growth and remodeling in response to adaptive and injury-related responses in the adult. In addition, control of normal vascular growth during maturation is likely to have redundant mechanisms.
Ischemia itself or the “inflammatory-like” factors that direct arteriogenesis (41) may augment norepinephrine release from vascular nerves. Balloon injury and ischemia induce norepinephrine release (9, 12). Also, conditions that stimulate growth of VSMCs and adventitial fibroblasts enhance their sensitivity to catecholamine trophic activity (45, 46, 52, 62). In ischemia, this may extend, in part, from the ability of reduced oxygen levels to increase expression of α1B-ARs by vascular wall cells (25, 26). Therefore, catecholamines may act to amplify growth and remodeling mechanisms important in arteriogenesis and angiogenesis.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-62584 (J. E. Faber) and T32-HL-069768 (J. A. Clayton).
We thank K. Kirk McNaughton for histology and Dr. Hyungsuk Kim for conducting the renal renin mRNA assay.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2005 by the American Physiological Society