Tissue Angiotensin-Converting Enzyme in Imposed and Physiological Flow-Related Arterial Remodeling in Mice
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《动脉硬化血栓血管生物学》
From the Department of Pharmacology & Toxicology (R.H.P.H, P.M.H.S., W.M.A., G.E.F., J.F.M.S, J.G.R.D.M.), Cardiovascular Research Institute Maastricht (CARIM), Universiteit Maastricht, Maastricht, the Netherlands; and the School of Life Sciences (G.E.F., J.G.R.D.M.), Transnational University of Limburg, Maastricht, the Netherlands.
ABSTRACT
Objective— To test whether membrane-bound angiotensin I-converting enzyme (t-ACE) is involved in arterial remodeling, we applied unilateral carotid artery (CA) ligation and studied uterine arteries (UA) before, during, and after pregnancy in t-ACE–/– and t-ACE+/+ mice.
Results— In CA of t-ACE–/– mice, blood pressure, outer diameter (D), and medial cross-sectional area (mCSA) were reduced, whereas blood flow (BF) and the number of medial cells (mC) were not modified. In the ligated CA, mCSA and number of mC were increased while outer D and distensibility were reduced. These changes were significantly less pronounced in t-ACE–/– than t-ACE+/+ mice. In UA of t-ACE–/– mice, D was larger and mCSA was unaltered. At term pregnancy, D and mCSA of the UA were reversibly increased. Structural changes of UA during and after pregnancy were comparable in both strains.
Conclusions— t-ACE contributes to arterial structure and remodeling. It plays a major role in hyperplastic inward remodeling of the CA imposed by blood flow cessation, but it is not essential for outward hypertrophic and subsequent inward hypotrophic remodeling of the UA during and after pregnancy.
Key Words: tissue ACE ? uterine artery ? carotid artery ? pregnancy ? flow-induced remodeling ? knockout mice
Introduction
Many components of the renin-angiotensin system are localized in tissues indicating the existence of a local renin-angiotensin system.1 Angiotensin-converting enzyme (ACE) is present in all major organs and blood vessels.2 This tissue-bound ACE (t-ACE) plays a major role in the local production of angiotensin II (Ang II)3 and in the local degradation of bradykinin.4
Ang II modulates vasomotor tone,5 cell growth and apoptosis,6,7 and cell migration and extracellular matrix deposition,8,9 and stimulates the production of other vasoconstrictor and growth factors.10 It plays important roles in the functional and structural integrity of the arterial wall and in the pathogenesis of cardiovascular diseases.11,12 Experimental and clinical studies with ACE inhibitors and angiotensin-1 (AT1) receptor antagonists consistently documented beneficial effects of these agents in treating and preventing cardiovascular diseases such as hypertension and atherosclerosis.13,14 Some of the beneficial effects of ACE inhibitors can be attributed to reduced breakdown of bradykinin. Antiproliferative effects of kinins via B2-receptors were demonstrated in arterial injury models using both ACE inhibitors and B2-receptor antagonists.15,16
Pressure and flow determine circumferential wall stress (CWS) and wall shear stress (WSS) in the arterial system. On an acute basis, they modulate arterial smooth muscle tone: an increase in transmural pressure triggers a myogenic contractile response;17 an increase in flow induces an endothelium-dependent vasodilatation.18 On a chronic basis, negative feedback control of CWS and WSS is achieved by modulation of arterial wall thickness and arterial lumen diameter, respectively.19,20 The mechanisms of this arterial remodeling largely remain to be established.21–23 They might involve Ang II that is produced within the arterial wall in response to an increase in CWS.19 Furthermore, there is increasing evidence that elevated WSS releases bradykinin from the endothelium.24,25
Arterial remodeling in response to altered blood flow is often investigated using surgery to shunt flow between adjacent arteries.20–23,26–30 For instance, on unilateral ligation of a carotid artery, the arterial blood flow is acutely and persistently abolished in the ipsilateral artery and doubled in the contralateral artery. Thereafter, the lumen diameter and wall mass of the vessels are modified and a neointima may develop.21,26–30 Also physiological challenges may be considered. During pregnancy, blood flow through the uterine circulation increases substantially and reversibly.31 In the mouse, the uterine vasculature undergoes luminal expansion and an increase in wall mass,25 to accommodate this increase in uterine blood flow. The relationships between imposed, physiological, and pathological arterial remodeling remain incompletely understood.
Two strains of ACE-deficient mice have been generated, 1 that completely lacks somatic ACE32 and 1 that lacks the carboxy-terminal membrane-spanning anchor region.33 In the latter, t-ACE-deficient (t-ACE–/–) mice, blood pressure is reduced33,34 and the structural and mechanical properties of the aorta, carotid artery, and mesenteric resistance artery are modified.34
In the present study, we hypothesized that membrane-bound ACE plays a pivotal role during flow-induced arterial remodeling. To test this hypothesis, we applied unilateral carotid artery ligations26 and studied uterine arteries before, during, and after pregnancy25 in wild-type and t-ACE–/– mice.
Methods
Animals
The generation of mice with C57Bl6/SV genetic background and lacking t-ACE (t-ACE–/–) has been described by Esther et al.32 Mice heterozygous for the mutated ACE allele (t-ACE±) were mated to obtain mice homozygous for the mutated allele (t-ACE–/–) and their wild-type littermates (t-ACE+/+). All experiments were conducted according to institutional guidelines. Genotyping was performed as previously described.34
Hemodynamics and Surgery
Mice were anesthetized with pentobarbital (10 mg/kg), fixed on a heating pad to control body temperature at 37°C, and blood pressure and heart rate were determined as described.34
At 3 to 4 months of age, unilateral carotid artery ligation was performed in male t-ACE+/+ (n=12) and t-ACE–/– (n=12) mice as previously described.21,26 One of the t-ACE+/+ and 2 of the t-ACE–/– mice died within 24 hours after the surgery. In sham-operated t-ACE+/+ and t-ACE–/– mice, right carotid arteries (RCAs) and left carotid arteries (LCAs) were exposed but not ligated.
Four weeks after surgery, the animals were again anesthetized. Blood flow was recorded in the LCA and RCA using an ultrasonic flow probe (0.5 mm V series; Transonic Systems) as previously described.21,25
Pregnancy-Induced Uterine Artery Remodeling
Female mice (age 4 to 6 months) were used to study uterine arterial remodeling, comparing t-ACE–/– with t-ACE+/+. In view of the infertility of male t-ACE–/– mice,32 male t-ACE+/+ mice were used for mating virgin (in the remainder of this article referred to as nonpregnant) t-ACE+/+ and t-ACE–/– mice. Uterine arteries were investigated before pregnancy (NP), at late pregnancy (LP; day 18 to 19), and at 7 days postpartum (PP).
Pressure-Diameter Curves
Carotid artery segments (5 mm long, halfway between the aortic arch and the carotid bifurcation) and uterine artery segments (3 mm long, halfway between the ovaries and the vagina) were isolated and transferred into an organ chamber (arteriograph system; Living System Instrumentation, Burlington, Vt). The chamber was filled with calcium-free HEPES buffer containing 10 μmol/L sodium nitroprusside to assure maximal vasodilatation. Because of the intransparancy of the carotid artery, outer diameters were recorded in the carotid artery, and internal diameters were recorded for the uterine artery. A pressure-diameter relationship was established by recording the diameter during step-wise (10 mm Hg) increases in intraluminal pressures from 20 to 150 mm Hg. After the experiment, the arteries were fixed at a transmural pressure of 100 mm Hg in 4% phosphate-buffered formaldehyde.
Morphometry
Fixed vessels were embedded in paraffin and cross-sections (4 μm) were stained with Lawson solution (Boom, Meppel, the Netherlands) to visualize the elastic laminae. In the case of carotid arteries, the morphometric analysis focused on the central part of the vessel, halfway between the aortic arch and the carotid bifurcation. Unlike the distal part26 near the ligation, the central part showed no signs of neointima formation or inflammation. Video images were made from cross-sections using a Zeiss axioscope and a standard charge-coupled device camera (Sony). Medial cross-sectional area, the average number of medial nuclear profiles per cross-section, and contents and densities of elastin and collagen were determined as described.34
Statistics
Results are expressed as means±SEM. Statistical significance of differences between sets of data were determined by ANOVA with post-hoc Student-Newman-Keuls test. P<0.05 was considered to indicate a significant difference.
Results
General Observations
Blood pressure was significantly lower in male t-ACE–/– mice compared with t-ACE+/+ mice (75±4 mm Hg versus 100±5 mm Hg), while heart rate was comparable (627±21 bpm versus 662±12 bpm, respectively). Carotid arterial blood flow (mean of LCA and RCA) did not differ significantly between t-ACE–/– and t-ACE+/+ (0.39±0.04 mL/min versus 0.40±0.02 mL/min, respectively). In both groups of mice, LCA ligation resulted in an elimination of blood flow in the occluded vessel and in a comparable substantial increase of blood flow in the RCA (0.81±0.11 mL/min in t-ACE–/– and 0.79±0.07 mL/min in t-ACE+/+).
In female mice, deficiency of t-ACE did not result in abnormalities during and after pregnancy as far as body weight, fetal and placental weight, and the number of fetuses and pups are concerned, but cardiac hypertrophy was reduced (Table I, available online at http://atvb.ahajournals.org).
Arterial Structure of t-ACE+/+ and t-ACE–/– Mice
Carotid artery outer diameters were significantly smaller in t-ACE–/– compared with tACE+/+ mice (Figure 1A). Medial cross-sectional area was significantly smaller in carotid artery of t-ACE–/– mice (14.1±0.8x103 μm2) than t-ACE+/+ mice (20.4±0.9x103 μm2). The density and content of collagen did not differ, but the density and content of elastin tended to be smaller in carotid artery of t-ACE–/– than t-ACE+/+ mice (Table II, available online at http://atvb.ahajournals.org).
Figure 1. A, Relations between distending intraluminal pressure and outer diameter in isolated carotid arteries of t-ACE+/+ (white symbols) and t-ACE–/– (black symbols) mice. Inset, Medial CSA of isolated carotid arteries of t-ACE+/+ (white bars) and t-ACE–/– (black bars) male mice. B, Relations between distending intraluminal pressure and inner diameter in isolated uterine arteries of nonpregnant t-ACE+/+ (white symbols) and t-ACE–/– (black symbols) mice. Inset, Medial CSA of isolated uterine arteries of nonpregnant t-ACE+/+ (white bars) and t-ACE–/– (black bars) mice. Values are means±SEM. *P<0.05 versus t-ACE+/+.
In contrast to carotid artery, uterine arteries of nonpregnant t-ACE–/– were significantly wider compared with t-ACE+/+ mice (Figure 1B), despite comparable medial cross-sectional area (CSA) (Figure 1B, inset).
Carotid Artery Structural Changes in Response to Altered Blood Flow
The hyperperfused RCA displayed a significant increase in outer diameter (Figure 2). At high transmural pressure, the diameter was increased in both strains. At low transmural pressure, the diameter was significantly increased in t-ACE–/– but not t-ACE+/+ mice. Medial collagen, elastin, and CSA were not significantly altered in the RCA of t-ACE–/– and t-ACE+/+ mice after exposure to elevated blood flow (Table II).
Figure 2. Effects of unilateral carotid artery ligation on relations between distending intraluminal pressure and outer carotid artery diameter in t-ACE+/+ (A) and t-ACE–/– (B) mice. Sham indicates sham-operated; NF, no-flow (ligated) vessels; HF, high-flow (hyperperfused) vessels. Values are means±SEM. #P<0.05 versus Sham.
Ligation of the LCA resulted in a significant reduction of the outer diameter (Figure 2) accompanied by an increase in medial CSA (Table II) and a dramatic reduction in distensibility of the ligated vessel. The diameter reduction was more pronounced in t-ACE+/+ mice (from 671±13 to 516±25 μm; 23% reduction) than in t-ACE–/– mice (from 625±16 to 515±45 μm; 18% reduction). Also, the medial hypertrophy was more pronounced in t-ACE+/+ mice (from 20.4±0.9 to 34.8±4.2x103 μm2, representing a 71% increase) than t-ACE–/– mice (from 14.1±0.8 to 18.1±2.3x103 μm2, representing a 28% increase) and involved a large increase in the number of medial cells in t-ACE+/+ (from 85±2 to 164±3/cross-section) but not t-ACE–/– mice (from 81±3 to 75±7/cross-section).
In the medial of the ligated LCA of both t-ACE–/– and t-ACE+/+ mice, staining for the monocyte/macrophage marker ED1 was rare (not shown). Collagen and elastin densities were not altered in the ligated vessels, but elastin content was significantly increased in t-ACE+/+ mice (Table II).
Uterine Artery Structure During and After Pregnancy
During late pregnancy, maximal lumen diameter of uterine arteries increased significantly in t-ACE+/+ (from 251±7 to 384±14 μm) and t-ACE–/– mice (from 290±10 to 384±16 μm, Figure 3). Medial CSA increased significantly and comparably during pregnancy in uterine arteries of both t-ACE+/+ (from 2.6±0.3 to 5.2±0.8x103 μm2) and t-ACE–/– mice (from 2.9±0.3 to 5.1±0.4x103 μm2, Figure I, available online at http://atvb.ahajournals.org). In neither strain was the medial hypertrophy accompanied by a significant change in the number of nuclear profiles per medial cross section (Figure I). By 7 days postpartum, most of the structural changes in the uterine artery were reversed (Figure 3). Maximal lumen diameter had partially regressed to prepregnant values in both t-ACE+/+ mice (from 384±14 to 290±21 μm) and t-ACE–/– mice (from 384±16 to 301±17 μm). Medial CSA regressed to nonpregnant values in both strains (Figure I).
Figure 3. Effects of pregnancy on the relations between distending intraluminal pressure and inner diameter in isolated uterine arteries of nonpregnant (NP), late pregnant (LP), and postpartum (PP) t-ACE+/+ (A) and t-ACE–/– (B) mice. #P<0.05 versus NP and PP. Values are means±SEM.
Discussion
Membrane-bound ACE participates in the control of arterial structure. The role of the enzyme differs between imposed and physiological arterial structural changes.
In line with the pressor and hypertrophic effects of angiotensin II,6,12,35 t-ACE–/– mice displayed lower blood pressure, reduced carotid artery medial mass, and reduced cardiac hypertrophy. As previously reported,34 the carotid artery was narrower but more distensible despite unaltered carotid arterial blood flow. Maintenance of carotid artery blood flow, despite reduced blood pressure, implies reduced cerebrovascular resistance. This is largely caused by blunted vasomotor tone and an increased structural diameter of resistance arteries as observed in the mesenteric34 and uterine arterial bed (this study). We previously observed that chronic pharmacological ACE inhibition in wild-type mice resulted in comparable alterations of carotid artery structure and mechanics.34 These observations indicate that t-ACE plays a role in the establishment and maintenance of the balance between local hemodynamic and mechanical forces, on the one hand, and the structural and mechanical properties of large elastic arteries, on the other hand. Because a 2-week treatment with a B2-receptor antagonist blunts the difference in carotid artery mass and distensibility, but not the difference in blood pressure between t-ACE–/– and t-ACE+/+ mice,34 primarily bradykinin seems to be involved.
In contrast to large elastic arteries, but similar to small muscular mesenteric arteries,34 uterine arteries of t-ACE–/– displayed a larger lumen diameter despite a comparable medial surface area. This regionality is reminiscent of the hypertrophy and inward eutrophic remodeling that was described for large elastic and small muscular arteries in hypertension, respectively36,37 It was proposed that negative feedback control of local mechanical forces can be achieved to a larger extent in muscular arteries than in elastic arteries by alterations in vasomotor tone, ie, by myogenic tone-induced and flow-induced vasodilatation.17,18
Local mechanical forces that govern arterial structure include CWS and WSS.17,18 Chronic changes of these forces result in arterial remodeling.20–23,25–30 These structural responses involve partly interrelated alterations in: (1) arterial wall mass; (2) mechanical properties of the arterial wall material; and (3) arterial lumen diameter. Because CWS and WSS stimulate intra-arterial production of angiotensin II and bradykinin, respectively,19,24,25 we compared arterial remodeling in large and small muscular arteries of t-ACE–/– and t-ACE+/+ mice. In the mouse carotid artery ligation model, no differences were observed between males and females.27
The increase in contralateral blood flow after unilateral carotid artery ligation, was comparable in t-ACE–/– and t-ACE+/+ mice. After 4 weeks of exposure to doubled blood flow, arterial diameter was increased whereas arterial wall mass and collagen and elastin content were not altered. Although t-ACE was not essential for this outward arterial remodeling, distinct ultrastructural alterations might have contributed to the expansion of the vessel. In the absence of t-ACE, which has been shown to be a determinant of large artery stiffness,34 not only the maximal diameter but also the diameters at intermediate pressures were increased.
In ligated carotid arteries of t-ACE+/+ mice, outer diameter and arterial distensibility were reduced while medial cross-sectional area and medial cell number were increased. The marked stiffening of the arterial wall proceeded without a change in collagen and elastin density; rather, the elastin content significantly increased. Changes in diameter, medial cross-sectional area and cell number were less pronounced in t-ACE–/– than t-ACE+/+ mice. These findings are in line with previously reported angiotensin II-induced stimulation of vascular smooth muscle cell proliferation and fibronectin production.9,12,19 Thus, t-ACE is involved in the structural response of large elastic arteries to a moderate increase and a marked decrease in blood flow.
In both t-ACE–/– and t-ACE+/+ mice, the increase in uterine blood flow during pregnancy was sufficient to support the survival and growth of a comparable number of fetuses. Diameter and medial mass of the uterine artery increased substantially during pregnancy. In t-ACE–/– mice, the diameter increase was somewhat smaller than in t-ACE+/+ mice. Uterine artery remodeling during pregnancy is not altered in tissue-kallikrein-deficient mice.25 Consequently, angiotensin II rather than bradykinin seems to be involved in the widening of the vessel. The increase in medial mass was comparable in both strains. Because the number of nuclear profiles per medial section did not change, the wall hypertrophy is primarily caused by cell growth. Unlike for the hyperplasia in the no-flow carotid artery, t-ACE–/– is not essential for cellular hypertrophy in the uterine artery.
By 7 days postpartum, uterine artery structure no longer differed from prepregnancy values. This illustrates the dynamic nature of the vascular bed. The reversal of the remodeling was obtained by a reduction of medial cell size. Also tissue-kallikrein-deficiency does not impair reversal of uterine artery remodeling.25 Consequently, intramural production of neither angiotensin II nor bradykinin are essential for the adjustments of uterine artery structure to the marked reduction in blood flow.
Not only the role of t-ACE but also the nature of the remodeling differed in carotid and uterine arteries. Hyperperfusion led to expansion of both vessels, but this involved wall and cellular hypertrophy in the uterine artery and not in the carotid artery. Hypoperfusion resulted in a reduction of lumen diameter in both vessels. This was accompanied by wall hypertrophy and hyperplasia in the carotid artery, whereas the number of medial cells was not modified and their size was reduced in the uterine artery. These regional differences seem caused by, but not solely by, the different extents and kinetics of the imposed and physiological blood flow changes. In rat and mouse small muscular mesenteric arteries, a doubling of blood flow leads to outward hypertrophic remodeling, and a marked reduction of blood flow leads to inward hypotrophic remodeling.20,23,38 Unlike in the uterine artery, these 2 types of mesenteric artery remodeling are accompanied by medial cell proliferation and apoptosis38 leading to a gain and a loss of smooth muscle cells in the high-flow and the low-flow arteries, respectively. Whether the role of t-ACE is more prominent in the remodeling of large elastic than in small muscular arteries, or is more prominent in hyperplastic than in hypertrophic remodeling, awaits further experimentation.
The regional heterogeneity and diversity of the role of t-ACE in arterial structural changes, which we observed in an elastic artery and a pre-existing collateral artery such as the uterine artery, are in line with earlier findings. Angiotensin II and bradykinin stimulate and inhibit the proliferation of medial and neointimal smooth muscle cells in carotid arteries, respectively.12,16 Yet, angiotensin II can promote shunting of blood flow through pre-existing collateral channels,39 and has been reported to promote and inhibit angiogenesis at low and high concentrations, respectively.40 The former mechanism, along with the proangiogenic effects of bradykinin,41 seems to account for the beneficial effects on capillary density and blood flow observed with inhibitors of t-ACE such as quinaprilat in peripheral and coronary ischemia.42
Despite its roles in the morphogenesis and remodeling of vascular beds and in spermatogenesis,32 t-ACE (this study) and even membrane-bound and circulating ACE33 are not essential for optimal pregnancy outcome in the mouse. The various maternal systemic and local vascular alterations, including extensive remodeling and angiogenesis in the uterine vascular bed, can proceed in the absence of ACE-derived angiotensin II and do not require local production of bradykinin.25 Alternative biosynthetic pathways and various other growth factors and angiogenic stimuli might be upregulated during this physiological response on which there is considerable biological pressure. Recent genetic observations indicate that ACE might rather be detrimental during human pregnancy.43 The ACE I/D polymorphism affects, besides ACE activity, uteroplacental and umbilical blood flows and the recurrence of an adverse pregnancy outcome, including intrauterine growth retardation, in women with a history of preeclampsia.
In summary, observations in t-ACE–/– mice indicate that membrane-bound ACE plays different roles in the development and maintenance of the structural and mechanical properties of large elastic and small muscular arteries. Moreover, our present findings in t-ACE–/– mice, combined with our earlier findings in tissue-kallikrein-deficient mice,25 indicate that intra-arterial production of angiotensin II contributes substantially to the hyperplastic remodeling of the ligated carotid artery but not to the hypertrophic and hypotrophic remodeling of the uterine artery during and after pregnancy.
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ABSTRACT
Objective— To test whether membrane-bound angiotensin I-converting enzyme (t-ACE) is involved in arterial remodeling, we applied unilateral carotid artery (CA) ligation and studied uterine arteries (UA) before, during, and after pregnancy in t-ACE–/– and t-ACE+/+ mice.
Results— In CA of t-ACE–/– mice, blood pressure, outer diameter (D), and medial cross-sectional area (mCSA) were reduced, whereas blood flow (BF) and the number of medial cells (mC) were not modified. In the ligated CA, mCSA and number of mC were increased while outer D and distensibility were reduced. These changes were significantly less pronounced in t-ACE–/– than t-ACE+/+ mice. In UA of t-ACE–/– mice, D was larger and mCSA was unaltered. At term pregnancy, D and mCSA of the UA were reversibly increased. Structural changes of UA during and after pregnancy were comparable in both strains.
Conclusions— t-ACE contributes to arterial structure and remodeling. It plays a major role in hyperplastic inward remodeling of the CA imposed by blood flow cessation, but it is not essential for outward hypertrophic and subsequent inward hypotrophic remodeling of the UA during and after pregnancy.
Key Words: tissue ACE ? uterine artery ? carotid artery ? pregnancy ? flow-induced remodeling ? knockout mice
Introduction
Many components of the renin-angiotensin system are localized in tissues indicating the existence of a local renin-angiotensin system.1 Angiotensin-converting enzyme (ACE) is present in all major organs and blood vessels.2 This tissue-bound ACE (t-ACE) plays a major role in the local production of angiotensin II (Ang II)3 and in the local degradation of bradykinin.4
Ang II modulates vasomotor tone,5 cell growth and apoptosis,6,7 and cell migration and extracellular matrix deposition,8,9 and stimulates the production of other vasoconstrictor and growth factors.10 It plays important roles in the functional and structural integrity of the arterial wall and in the pathogenesis of cardiovascular diseases.11,12 Experimental and clinical studies with ACE inhibitors and angiotensin-1 (AT1) receptor antagonists consistently documented beneficial effects of these agents in treating and preventing cardiovascular diseases such as hypertension and atherosclerosis.13,14 Some of the beneficial effects of ACE inhibitors can be attributed to reduced breakdown of bradykinin. Antiproliferative effects of kinins via B2-receptors were demonstrated in arterial injury models using both ACE inhibitors and B2-receptor antagonists.15,16
Pressure and flow determine circumferential wall stress (CWS) and wall shear stress (WSS) in the arterial system. On an acute basis, they modulate arterial smooth muscle tone: an increase in transmural pressure triggers a myogenic contractile response;17 an increase in flow induces an endothelium-dependent vasodilatation.18 On a chronic basis, negative feedback control of CWS and WSS is achieved by modulation of arterial wall thickness and arterial lumen diameter, respectively.19,20 The mechanisms of this arterial remodeling largely remain to be established.21–23 They might involve Ang II that is produced within the arterial wall in response to an increase in CWS.19 Furthermore, there is increasing evidence that elevated WSS releases bradykinin from the endothelium.24,25
Arterial remodeling in response to altered blood flow is often investigated using surgery to shunt flow between adjacent arteries.20–23,26–30 For instance, on unilateral ligation of a carotid artery, the arterial blood flow is acutely and persistently abolished in the ipsilateral artery and doubled in the contralateral artery. Thereafter, the lumen diameter and wall mass of the vessels are modified and a neointima may develop.21,26–30 Also physiological challenges may be considered. During pregnancy, blood flow through the uterine circulation increases substantially and reversibly.31 In the mouse, the uterine vasculature undergoes luminal expansion and an increase in wall mass,25 to accommodate this increase in uterine blood flow. The relationships between imposed, physiological, and pathological arterial remodeling remain incompletely understood.
Two strains of ACE-deficient mice have been generated, 1 that completely lacks somatic ACE32 and 1 that lacks the carboxy-terminal membrane-spanning anchor region.33 In the latter, t-ACE-deficient (t-ACE–/–) mice, blood pressure is reduced33,34 and the structural and mechanical properties of the aorta, carotid artery, and mesenteric resistance artery are modified.34
In the present study, we hypothesized that membrane-bound ACE plays a pivotal role during flow-induced arterial remodeling. To test this hypothesis, we applied unilateral carotid artery ligations26 and studied uterine arteries before, during, and after pregnancy25 in wild-type and t-ACE–/– mice.
Methods
Animals
The generation of mice with C57Bl6/SV genetic background and lacking t-ACE (t-ACE–/–) has been described by Esther et al.32 Mice heterozygous for the mutated ACE allele (t-ACE±) were mated to obtain mice homozygous for the mutated allele (t-ACE–/–) and their wild-type littermates (t-ACE+/+). All experiments were conducted according to institutional guidelines. Genotyping was performed as previously described.34
Hemodynamics and Surgery
Mice were anesthetized with pentobarbital (10 mg/kg), fixed on a heating pad to control body temperature at 37°C, and blood pressure and heart rate were determined as described.34
At 3 to 4 months of age, unilateral carotid artery ligation was performed in male t-ACE+/+ (n=12) and t-ACE–/– (n=12) mice as previously described.21,26 One of the t-ACE+/+ and 2 of the t-ACE–/– mice died within 24 hours after the surgery. In sham-operated t-ACE+/+ and t-ACE–/– mice, right carotid arteries (RCAs) and left carotid arteries (LCAs) were exposed but not ligated.
Four weeks after surgery, the animals were again anesthetized. Blood flow was recorded in the LCA and RCA using an ultrasonic flow probe (0.5 mm V series; Transonic Systems) as previously described.21,25
Pregnancy-Induced Uterine Artery Remodeling
Female mice (age 4 to 6 months) were used to study uterine arterial remodeling, comparing t-ACE–/– with t-ACE+/+. In view of the infertility of male t-ACE–/– mice,32 male t-ACE+/+ mice were used for mating virgin (in the remainder of this article referred to as nonpregnant) t-ACE+/+ and t-ACE–/– mice. Uterine arteries were investigated before pregnancy (NP), at late pregnancy (LP; day 18 to 19), and at 7 days postpartum (PP).
Pressure-Diameter Curves
Carotid artery segments (5 mm long, halfway between the aortic arch and the carotid bifurcation) and uterine artery segments (3 mm long, halfway between the ovaries and the vagina) were isolated and transferred into an organ chamber (arteriograph system; Living System Instrumentation, Burlington, Vt). The chamber was filled with calcium-free HEPES buffer containing 10 μmol/L sodium nitroprusside to assure maximal vasodilatation. Because of the intransparancy of the carotid artery, outer diameters were recorded in the carotid artery, and internal diameters were recorded for the uterine artery. A pressure-diameter relationship was established by recording the diameter during step-wise (10 mm Hg) increases in intraluminal pressures from 20 to 150 mm Hg. After the experiment, the arteries were fixed at a transmural pressure of 100 mm Hg in 4% phosphate-buffered formaldehyde.
Morphometry
Fixed vessels were embedded in paraffin and cross-sections (4 μm) were stained with Lawson solution (Boom, Meppel, the Netherlands) to visualize the elastic laminae. In the case of carotid arteries, the morphometric analysis focused on the central part of the vessel, halfway between the aortic arch and the carotid bifurcation. Unlike the distal part26 near the ligation, the central part showed no signs of neointima formation or inflammation. Video images were made from cross-sections using a Zeiss axioscope and a standard charge-coupled device camera (Sony). Medial cross-sectional area, the average number of medial nuclear profiles per cross-section, and contents and densities of elastin and collagen were determined as described.34
Statistics
Results are expressed as means±SEM. Statistical significance of differences between sets of data were determined by ANOVA with post-hoc Student-Newman-Keuls test. P<0.05 was considered to indicate a significant difference.
Results
General Observations
Blood pressure was significantly lower in male t-ACE–/– mice compared with t-ACE+/+ mice (75±4 mm Hg versus 100±5 mm Hg), while heart rate was comparable (627±21 bpm versus 662±12 bpm, respectively). Carotid arterial blood flow (mean of LCA and RCA) did not differ significantly between t-ACE–/– and t-ACE+/+ (0.39±0.04 mL/min versus 0.40±0.02 mL/min, respectively). In both groups of mice, LCA ligation resulted in an elimination of blood flow in the occluded vessel and in a comparable substantial increase of blood flow in the RCA (0.81±0.11 mL/min in t-ACE–/– and 0.79±0.07 mL/min in t-ACE+/+).
In female mice, deficiency of t-ACE did not result in abnormalities during and after pregnancy as far as body weight, fetal and placental weight, and the number of fetuses and pups are concerned, but cardiac hypertrophy was reduced (Table I, available online at http://atvb.ahajournals.org).
Arterial Structure of t-ACE+/+ and t-ACE–/– Mice
Carotid artery outer diameters were significantly smaller in t-ACE–/– compared with tACE+/+ mice (Figure 1A). Medial cross-sectional area was significantly smaller in carotid artery of t-ACE–/– mice (14.1±0.8x103 μm2) than t-ACE+/+ mice (20.4±0.9x103 μm2). The density and content of collagen did not differ, but the density and content of elastin tended to be smaller in carotid artery of t-ACE–/– than t-ACE+/+ mice (Table II, available online at http://atvb.ahajournals.org).
Figure 1. A, Relations between distending intraluminal pressure and outer diameter in isolated carotid arteries of t-ACE+/+ (white symbols) and t-ACE–/– (black symbols) mice. Inset, Medial CSA of isolated carotid arteries of t-ACE+/+ (white bars) and t-ACE–/– (black bars) male mice. B, Relations between distending intraluminal pressure and inner diameter in isolated uterine arteries of nonpregnant t-ACE+/+ (white symbols) and t-ACE–/– (black symbols) mice. Inset, Medial CSA of isolated uterine arteries of nonpregnant t-ACE+/+ (white bars) and t-ACE–/– (black bars) mice. Values are means±SEM. *P<0.05 versus t-ACE+/+.
In contrast to carotid artery, uterine arteries of nonpregnant t-ACE–/– were significantly wider compared with t-ACE+/+ mice (Figure 1B), despite comparable medial cross-sectional area (CSA) (Figure 1B, inset).
Carotid Artery Structural Changes in Response to Altered Blood Flow
The hyperperfused RCA displayed a significant increase in outer diameter (Figure 2). At high transmural pressure, the diameter was increased in both strains. At low transmural pressure, the diameter was significantly increased in t-ACE–/– but not t-ACE+/+ mice. Medial collagen, elastin, and CSA were not significantly altered in the RCA of t-ACE–/– and t-ACE+/+ mice after exposure to elevated blood flow (Table II).
Figure 2. Effects of unilateral carotid artery ligation on relations between distending intraluminal pressure and outer carotid artery diameter in t-ACE+/+ (A) and t-ACE–/– (B) mice. Sham indicates sham-operated; NF, no-flow (ligated) vessels; HF, high-flow (hyperperfused) vessels. Values are means±SEM. #P<0.05 versus Sham.
Ligation of the LCA resulted in a significant reduction of the outer diameter (Figure 2) accompanied by an increase in medial CSA (Table II) and a dramatic reduction in distensibility of the ligated vessel. The diameter reduction was more pronounced in t-ACE+/+ mice (from 671±13 to 516±25 μm; 23% reduction) than in t-ACE–/– mice (from 625±16 to 515±45 μm; 18% reduction). Also, the medial hypertrophy was more pronounced in t-ACE+/+ mice (from 20.4±0.9 to 34.8±4.2x103 μm2, representing a 71% increase) than t-ACE–/– mice (from 14.1±0.8 to 18.1±2.3x103 μm2, representing a 28% increase) and involved a large increase in the number of medial cells in t-ACE+/+ (from 85±2 to 164±3/cross-section) but not t-ACE–/– mice (from 81±3 to 75±7/cross-section).
In the medial of the ligated LCA of both t-ACE–/– and t-ACE+/+ mice, staining for the monocyte/macrophage marker ED1 was rare (not shown). Collagen and elastin densities were not altered in the ligated vessels, but elastin content was significantly increased in t-ACE+/+ mice (Table II).
Uterine Artery Structure During and After Pregnancy
During late pregnancy, maximal lumen diameter of uterine arteries increased significantly in t-ACE+/+ (from 251±7 to 384±14 μm) and t-ACE–/– mice (from 290±10 to 384±16 μm, Figure 3). Medial CSA increased significantly and comparably during pregnancy in uterine arteries of both t-ACE+/+ (from 2.6±0.3 to 5.2±0.8x103 μm2) and t-ACE–/– mice (from 2.9±0.3 to 5.1±0.4x103 μm2, Figure I, available online at http://atvb.ahajournals.org). In neither strain was the medial hypertrophy accompanied by a significant change in the number of nuclear profiles per medial cross section (Figure I). By 7 days postpartum, most of the structural changes in the uterine artery were reversed (Figure 3). Maximal lumen diameter had partially regressed to prepregnant values in both t-ACE+/+ mice (from 384±14 to 290±21 μm) and t-ACE–/– mice (from 384±16 to 301±17 μm). Medial CSA regressed to nonpregnant values in both strains (Figure I).
Figure 3. Effects of pregnancy on the relations between distending intraluminal pressure and inner diameter in isolated uterine arteries of nonpregnant (NP), late pregnant (LP), and postpartum (PP) t-ACE+/+ (A) and t-ACE–/– (B) mice. #P<0.05 versus NP and PP. Values are means±SEM.
Discussion
Membrane-bound ACE participates in the control of arterial structure. The role of the enzyme differs between imposed and physiological arterial structural changes.
In line with the pressor and hypertrophic effects of angiotensin II,6,12,35 t-ACE–/– mice displayed lower blood pressure, reduced carotid artery medial mass, and reduced cardiac hypertrophy. As previously reported,34 the carotid artery was narrower but more distensible despite unaltered carotid arterial blood flow. Maintenance of carotid artery blood flow, despite reduced blood pressure, implies reduced cerebrovascular resistance. This is largely caused by blunted vasomotor tone and an increased structural diameter of resistance arteries as observed in the mesenteric34 and uterine arterial bed (this study). We previously observed that chronic pharmacological ACE inhibition in wild-type mice resulted in comparable alterations of carotid artery structure and mechanics.34 These observations indicate that t-ACE plays a role in the establishment and maintenance of the balance between local hemodynamic and mechanical forces, on the one hand, and the structural and mechanical properties of large elastic arteries, on the other hand. Because a 2-week treatment with a B2-receptor antagonist blunts the difference in carotid artery mass and distensibility, but not the difference in blood pressure between t-ACE–/– and t-ACE+/+ mice,34 primarily bradykinin seems to be involved.
In contrast to large elastic arteries, but similar to small muscular mesenteric arteries,34 uterine arteries of t-ACE–/– displayed a larger lumen diameter despite a comparable medial surface area. This regionality is reminiscent of the hypertrophy and inward eutrophic remodeling that was described for large elastic and small muscular arteries in hypertension, respectively36,37 It was proposed that negative feedback control of local mechanical forces can be achieved to a larger extent in muscular arteries than in elastic arteries by alterations in vasomotor tone, ie, by myogenic tone-induced and flow-induced vasodilatation.17,18
Local mechanical forces that govern arterial structure include CWS and WSS.17,18 Chronic changes of these forces result in arterial remodeling.20–23,25–30 These structural responses involve partly interrelated alterations in: (1) arterial wall mass; (2) mechanical properties of the arterial wall material; and (3) arterial lumen diameter. Because CWS and WSS stimulate intra-arterial production of angiotensin II and bradykinin, respectively,19,24,25 we compared arterial remodeling in large and small muscular arteries of t-ACE–/– and t-ACE+/+ mice. In the mouse carotid artery ligation model, no differences were observed between males and females.27
The increase in contralateral blood flow after unilateral carotid artery ligation, was comparable in t-ACE–/– and t-ACE+/+ mice. After 4 weeks of exposure to doubled blood flow, arterial diameter was increased whereas arterial wall mass and collagen and elastin content were not altered. Although t-ACE was not essential for this outward arterial remodeling, distinct ultrastructural alterations might have contributed to the expansion of the vessel. In the absence of t-ACE, which has been shown to be a determinant of large artery stiffness,34 not only the maximal diameter but also the diameters at intermediate pressures were increased.
In ligated carotid arteries of t-ACE+/+ mice, outer diameter and arterial distensibility were reduced while medial cross-sectional area and medial cell number were increased. The marked stiffening of the arterial wall proceeded without a change in collagen and elastin density; rather, the elastin content significantly increased. Changes in diameter, medial cross-sectional area and cell number were less pronounced in t-ACE–/– than t-ACE+/+ mice. These findings are in line with previously reported angiotensin II-induced stimulation of vascular smooth muscle cell proliferation and fibronectin production.9,12,19 Thus, t-ACE is involved in the structural response of large elastic arteries to a moderate increase and a marked decrease in blood flow.
In both t-ACE–/– and t-ACE+/+ mice, the increase in uterine blood flow during pregnancy was sufficient to support the survival and growth of a comparable number of fetuses. Diameter and medial mass of the uterine artery increased substantially during pregnancy. In t-ACE–/– mice, the diameter increase was somewhat smaller than in t-ACE+/+ mice. Uterine artery remodeling during pregnancy is not altered in tissue-kallikrein-deficient mice.25 Consequently, angiotensin II rather than bradykinin seems to be involved in the widening of the vessel. The increase in medial mass was comparable in both strains. Because the number of nuclear profiles per medial section did not change, the wall hypertrophy is primarily caused by cell growth. Unlike for the hyperplasia in the no-flow carotid artery, t-ACE–/– is not essential for cellular hypertrophy in the uterine artery.
By 7 days postpartum, uterine artery structure no longer differed from prepregnancy values. This illustrates the dynamic nature of the vascular bed. The reversal of the remodeling was obtained by a reduction of medial cell size. Also tissue-kallikrein-deficiency does not impair reversal of uterine artery remodeling.25 Consequently, intramural production of neither angiotensin II nor bradykinin are essential for the adjustments of uterine artery structure to the marked reduction in blood flow.
Not only the role of t-ACE but also the nature of the remodeling differed in carotid and uterine arteries. Hyperperfusion led to expansion of both vessels, but this involved wall and cellular hypertrophy in the uterine artery and not in the carotid artery. Hypoperfusion resulted in a reduction of lumen diameter in both vessels. This was accompanied by wall hypertrophy and hyperplasia in the carotid artery, whereas the number of medial cells was not modified and their size was reduced in the uterine artery. These regional differences seem caused by, but not solely by, the different extents and kinetics of the imposed and physiological blood flow changes. In rat and mouse small muscular mesenteric arteries, a doubling of blood flow leads to outward hypertrophic remodeling, and a marked reduction of blood flow leads to inward hypotrophic remodeling.20,23,38 Unlike in the uterine artery, these 2 types of mesenteric artery remodeling are accompanied by medial cell proliferation and apoptosis38 leading to a gain and a loss of smooth muscle cells in the high-flow and the low-flow arteries, respectively. Whether the role of t-ACE is more prominent in the remodeling of large elastic than in small muscular arteries, or is more prominent in hyperplastic than in hypertrophic remodeling, awaits further experimentation.
The regional heterogeneity and diversity of the role of t-ACE in arterial structural changes, which we observed in an elastic artery and a pre-existing collateral artery such as the uterine artery, are in line with earlier findings. Angiotensin II and bradykinin stimulate and inhibit the proliferation of medial and neointimal smooth muscle cells in carotid arteries, respectively.12,16 Yet, angiotensin II can promote shunting of blood flow through pre-existing collateral channels,39 and has been reported to promote and inhibit angiogenesis at low and high concentrations, respectively.40 The former mechanism, along with the proangiogenic effects of bradykinin,41 seems to account for the beneficial effects on capillary density and blood flow observed with inhibitors of t-ACE such as quinaprilat in peripheral and coronary ischemia.42
Despite its roles in the morphogenesis and remodeling of vascular beds and in spermatogenesis,32 t-ACE (this study) and even membrane-bound and circulating ACE33 are not essential for optimal pregnancy outcome in the mouse. The various maternal systemic and local vascular alterations, including extensive remodeling and angiogenesis in the uterine vascular bed, can proceed in the absence of ACE-derived angiotensin II and do not require local production of bradykinin.25 Alternative biosynthetic pathways and various other growth factors and angiogenic stimuli might be upregulated during this physiological response on which there is considerable biological pressure. Recent genetic observations indicate that ACE might rather be detrimental during human pregnancy.43 The ACE I/D polymorphism affects, besides ACE activity, uteroplacental and umbilical blood flows and the recurrence of an adverse pregnancy outcome, including intrauterine growth retardation, in women with a history of preeclampsia.
In summary, observations in t-ACE–/– mice indicate that membrane-bound ACE plays different roles in the development and maintenance of the structural and mechanical properties of large elastic and small muscular arteries. Moreover, our present findings in t-ACE–/– mice, combined with our earlier findings in tissue-kallikrein-deficient mice,25 indicate that intra-arterial production of angiotensin II contributes substantially to the hyperplastic remodeling of the ligated carotid artery but not to the hypertrophic and hypotrophic remodeling of the uterine artery during and after pregnancy.
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