Excessive Microvascular Adaptation to Changes in Blood Flow in Mice Lacking Gene Encoding for Desmin
Objective— Desmin, an intermediate filament, has a key role in the integrity of myocytes, and its absence induces cardiomyopathies. Mice lacking desmin (Des−/− group) exhibit microvascular dysfunction leading to smooth muscle hyporeactivity. We investigated the effect of the absence of desmin in mice (Des−/− mice versus Des+/+ mice) on the adaptation of mesenteric arteries to changes in blood flow.
Methods and Results— With the use of selective ligations of second-order mesenteric arteries, blood flow was either diminished (low flow [LF]) or elevated (high flow [HF]); respective LF to HF values were 136±18 to 206±29 μL/min for Des+/+ mice and 119±14 to 189±24 μL/min for Des−/−mice in daughter arteries. Two weeks after ligation, arteries were mounted in an arteriograph, allowing the measurement of diameter under controlled conditions of pressure and flow. In HF arteries, diameter changes in response to increases in pressure were higher in Des−/− mice than in Des+/+ mice. Conversely, in LF arteries, diameter was lower in Des−/− mice. Flow-dependent dilation was higher in HF arteries and lower in LF arteries than in control arteries. This adaptation was lower in Des−/− mice than in Des+/+ mice (11.6±3.1% versus 25.5±4.8% dilation, respectively). Endothelial NO synthase expression increased in HF arteries in both strains.
Conclusions— These findings provide a demonstration of the role of the intermediate filament desmin in microvascular remodeling. This dysfunction might take place in desmin-related myopathies.
Desmin is a constitutive subunit of the intermediate filaments in skeletal, cardiac, and smooth muscle cells.1 The absence of desmin or its disassembly is associated with skeletal and cardiac myopathies in mice and humans.2,3⇓ We have recently reported that mice lacking the gene encoding for desmin suffer from a selective microvascular disorder consisting of decreased reactivity of smooth muscle cells to vasodilator and vasoconstrictor stimuli.4 This defect in smooth muscle cell function might be related to decreased mitochondrial function and energy supply in cells of desmin-deficient (Des−/−) mice,5 which might impair both dilatory and contractile functions requiring phosphorylation. Indeed, protein kinase C binds to intermediate filaments, and the absence of desmin might affect protein kinase C–dependent functions. Similarly, calcium handling,6 potassium channel activity,7 and phospholipase activity depend on cytoskeletal integrity.8
The role of desmin in smooth muscle cell function motivates the hypothesis that its absence may affect the long-term functional and/or structural adaptation of resistance arteries to chronic changes in blood flow. Long-term adaptation of blood vessels to changes in pressure and/or blood flow allows optimal feeding of organs in physiological and pathological situations, including physical exercise, hypertension, diabetes, ischemic disease, and tumor growth. Thus, we assessed the adaptation of small (resistance) arteries to a 2-week change in blood flow. Whereas the adaptation of large arteries to chronic changes in blood flow has been widely studied,9,10⇓ the equivalent interpretation in resistance arteries remains sparse and controversial. In large arteries, the adaptation or remodeling induced by an increase in flow depends mainly on the endothelial production of NO.9,11⇓ In small resistance arteries, chronic exercise leads to an increased diameter and enhancement of endothelial NO and prostanoid production.12 In other studies, mesenteric arteries were alternatively ligated so that arteries from the same vascular bed had either low flow (LF) or high flow (HF).13,14⇓ In this model, flow changes without significant changes in blood pressure. In addition, this model allows the study of arteries subjected to LF, which is a situation representative of ischemic disease.
Although cytoskeletal proteins play a central role in endothelial structural and morphological responses to flow,15 no study to date has investigated the role of cytoskeletal proteins in a perfused and functional resistance artery submitted to physiological flow conditions. In the present study, we tested the hypothesis that microvascular adaptation to changes in blood flow might be modified by the absence of desmin. We used small mesenteric resistance arteries in mice lacking the gene encoding for desmin. Mesenteric arteries were submitted to selective ligations, as previously described,13,14⇓ to generate consecutive arteries with a normal flow (NF), HF, or LF rate within the same arterial bed.
Adult male littermate mice (Des−/− and Des+/+, 3 to 4 months old) were produced in Prof Denise Paulin’s laboratory5 (University of Paris). Surgery was performed to modify blood flow in the mesenteric arteries of mice as previously described in rats.13,14⇓ Briefly, a ligation of the second-order arterial and venous branches was performed near the gut (please see online Figure I, which is available at http://atvb.ahajournals.org). Ligation was performed with 7-0 silk surgical thread and was applied on every other first-order mesenteric artery side branch. Similar surgery, but without the ligation, was performed in the sham-operated group. Arteries were designed as LF, HF, NF, or sham (arteries from sham-operated mice).
All procedures involving the care and euthanasia of the study animals were in accordance with the European Community standards on the care and use of laboratory animals (Ministère de l’Agriculture, France, authorization No. 00577).
Although 112 mice were included in the present study, 204 mice were subjected to surgery, and 92 died from intestinal occlusion. The survival rate was 52% in Des−/− mice and 57% in Des+/+ mice.
Blood Pressure and Blood Flow Measurements
In mice anesthetized with pentobarbital (50 mg/kg IP), the right carotid artery was cannulated to measure blood pressure, as previously described.16,17⇓ A medial laparotomy was then performed. Body temperature was maintained at 37.5°C by a thermostatically controlled heating platform. A section of the ileum was extracted and spread over a gauze swab that had been dampened with a sterile physiological salt solution (PSS). A segment of a first-order mesenteric arterial side branch was dissected free of fat and connective tissue under a dissection microscope. With the use of a micromanipulator, a transit-time ultrasonic flow probe (0.5-mm V series, Transonic Systems,) was placed around the artery. Flow was determined with a T106 flowmeter (Transonic). A zero-flow reading was obtained by softly clamping the artery under investigation. Then, flow was determined and recorded over a period of 10 minutes (each flow value was the average of at least 3 minutes of recording).
Pressure Myograph Experiments
Segments of arteries were isolated from the mesenteric circulation and cannulated at both ends in a video-monitored perfusion system (LSI), as previously described.16–18⇓⇓ Briefly, arteries were bathed in PSS (pH 7.4, Po2 160 mm Hg, and Pco2 37 mm Hg). Pressure was controlled by a servo-perfusion system, and flow was generated by a peristaltic pump. Diameter changes, wall thickness, and pressure were measured continuously. At the end of each experiment, arteries were perfused and superfused with a calcium-free PSS containing EGTA (2 mmol/L) and sodium nitroprusside (SNP, 10 μmol/L), and pressure steps were repeated to determine the arterial passive diameter.4,16–18⇓⇓⇓
Histomorphometry of Isolated Resistance Arteries
Arteries were mounted in an arteriograph, as described above, and bathed in a calcium-free PSS containing EGTA (2 mmol/L) and SNP (10 μmol/L). Pressure was set at 75 mm Hg, and vessels were fixed in 10% formaldehyde in saline solution (30 minutes) and sectioned (10-μm-thick sections). Morphometric analysis was performed with an automated image processor.19
Changes in Diameter Over Time
In a preliminary group of experiments (48 mice), surgery was performed to alter blood flow, and mice were used 1, 2, or 3 weeks later (n=8 per group). Similarly, sham-operated mice were used 1, 2, or 3 weeks later (n=8 per group). These experiments were performed to determine the time course of the flow-induced remodeling in mouse mesenteric arteries, because previous studies using this model were conducted in different species. In each animal, a segment of mesenteric artery was mounted in a pressure myograph, and passive arterial diameter was determined for pressure values ranging from 10 to 150 mm Hg.4,16–18⇓⇓⇓
Blood Flow and Passive Diameter Measurements
In 16 Des−/− and 16 Des+/+ mice, surgery was performed to alter blood flow in mesenteric arteries. The experimental and sham-operated (control) groups (4 groups of 8 mice each) were used 2 weeks later to determine mean arterial blood pressure and blood flow in the mesenteric arteries. Arteries were then excised and cut into 2 segments. One segment was mounted in a pressure myograph, and passive arterial diameter was determined. From internal diameter values, cross-sectional compliance was calculated.4,17⇓ The other segment was also mounted in a pressure myograph and fixed at a pressure of 75 mm Hg for histomorphometric analysis.19
Because no difference was found in blood flow (Table) and passive diameter (online Figure I) between mice subjected to ligation and sham-operated mice, the following experiments were conducted without sham-operated mice. HF and LF arteries were compared with NF arteries from the same mouse.
Flow-Dependent Dilation and Pharmacological Study
In 20 other mice (n=10 per group), surgery was performed, and after 2 weeks of experimental conditions, arteries (HF, NF, and LF) were excised and subsequently cut into 2 segments. One segment was mounted in a pressure myograph. Pressure was set at 75 mm Hg, and myogenic tone developed.16,17⇓ Flow was then increased in steps, and the arterial dilation was determined.16,17⇓ Flow-mediated dilation was subsequently repeated after the addition of NG-nitro-l-arginine methyl ester (L-NAME, 0.1 mmol/L) to the bath.16,17⇓ With the other arterial segment, cumulative concentration-response curves to phenylephrine and calcium (in a calcium-free PSS containing 80 mmol/L KCl) were obtained. SNP concentration-response curves were then obtained after preconstriction of the arteries with phenylephrine (1 μmol/L).16,17⇓
Western Blot Analysis of Endothelial NO Synthesis
In a separate series of experiments involving 12 mice, surgery was performed as described above, and arteries were collected after 2 weeks (n=6 per group). As previously described,20,21⇓ arterial tissues were homogenized, and proteins were separated by SDS-PAGE (Mini gel protean II system [Bio-Rad], 100 V, with use of 300 mL 25 mmol/L Tris, 192 mmol/L glycine, and 0.1% SDS) with a 4% stacking gel followed by a 7% running gel. After migration (10 μg proteins per line), proteins were transferred (50 V overnight at 4°C, with use of 800 mL of 25 mmol/L Tris, 192 mmol/L glycine, and 10% methanol) to polyvinylidene difluoride blotting membranes (Immobilon-P, Millipore). Membranes were then washed in TBS-T buffer (containing 10 mmol/L Tris/base pH 7.5, 0.1 mol/L NaCl, 1 mmol/L EDTA, and 0.1% Tween 20) and blocked for 2 hours at room temperature (5% fat-free dry milk in TBS-T). Membranes were incubated for 90 minutes at room temperature with the primary antibody (anti–endothelial NO synthase [anti-eNOS], dilution 1:500 in TBS-T, Santa-Cruz Biotechnology, or anti-actin, dilution 1:500, Santa-Cruz Biotechnology), washed again (3 times for 10 minutes), and incubated with horseradish peroxidase–conjugated secondary antibody (1:2000, 90 minutes at room temperature, Santa Cruz). Membranes were washed (3 times for 10 minutes), and eNOS or actin was visualized by using an ECL-Plus Chemiluminescence kit (Amersham).
Results were expressed as mean±SEM. Significance of the differences among groups was determined by ANOVA (1-factor ANOVA or ANOVA for consecutive measurements). Means were compared by a paired t test or by the Bonferroni test for multigroup comparisons. Values of P<0.05 were considered significant.
Body weight and mean arterial blood pressure were not significantly affected by the surgical intervention in either Des+/+ or Des−/− mice compared with sham-operated mice (Table). Similarly, body weight and mean arterial blood pressure were not affected by the absence of desmin (Table).
A preliminary set of experiments was conducted in control mice to determine the time necessary for complete remodeling. The increase in diameter in HF arteries was significant 2 weeks after ligation. After 3 weeks, diameter in HF arteries was equivalent to that measured after 2 weeks (online Figure I). In LF arteries, the reduction in diameter was significant after 1 week and did not change thereafter (online Figure I). There was no significant difference between NF arterial diameter in mice subjected to arterial ligation and arterial diameter in sham-operated mice (online Figure I). Similarly, there was no significant difference between NF arterial diameter in mice subjected to arterial ligation and arterial diameter in sham-operated mice after 1, 2, or 3 weeks (data not shown). Finally, arterial diameter was not affected by the duration of the experiment (1 to 3 weeks) in NF arteries or in arteries from sham-operated mice.
Blood flow was measured in Des−/− and in Des+/+ mice. Blood flow was significantly higher in HF than in NF arteries. This increase was similar in Des−/− and Des+/+ mice. In LF arteries, flow rate was too low to be determined, although flow was visible. Blood flow in sham-operated mice was not altered in either strain (Table).
Passive arterial diameter was measured in Des−/− and Des+/+ mice (Figure 1). Passive diameter was higher in HF than in NF arteries. Conversely, it was lower in LF arteries than in NF arteries. The difference in diameter between HF and NF arteries was higher in Des−/− than in Des+/+ mice (eg, 32±4% increase in diameter in Des−/− mice versus 21±4% increase in Des+/+ mice when pressure was 100 mm Hg). In LF arteries, passive diameter was lower in Des−/− mice than in Des+/+ mice (Figure 1). The absence of desmin had no significant effect on the arterial diameter in NF arteries. There was no difference in arterial diameter between NF arteries and arteries isolated from sham-operated mice (eg, for a pressure of 100 mm Hg, 180±6 versus 175±8 μm, respectively, in Des+/+ mice and 171±9 versus 177±10 μm, respectively, in Des−/− mice; n=8 per group).
Wall thickness was measured in mesenteric arteries for pressure values ranging from 10 to 150 mm Hg (Figure 2A). Wall thickness was not significantly different in HF arteries versus NF arteries in either Des−/− or Des+/+ mice. Arterial wall thickness was higher in LF arteries than in NF arteries in Des−/− and Des+/+ mice, although this was significant only in Des+/+ mice (Figure 2A). Arterial wall cross-sectional compliance was markedly decreased in LF arteries, and this decrease was significantly more pronounced in Des−/− mice (Figure 2B). In HF arteries, arterial wall cross-sectional compliance was not significantly affected, although it slightly increased in Des−/− and Des+/+ mice (Figure 2B). The absence of desmin did not significantly change arterial wall thickness or arterial wall cross-sectional compliance in NF arteries (Figure 2A and 2B).
Medial thickness and wall-to-lumen ratio were also determined in these arteries after formaldehyde fixation at a pressure of 75 mm Hg. Medial thickness was not significantly different in NF arteries versus HF arteries and was significantly decreased in LF arteries (Figure 2C). The wall-to-lumen ratio was increased in LF arteries compared with NF arteries, whereas it was not affected in HF arteries (Figure 2D). There was no significant difference in medial thickness and the wall-to-lumen ratio between Des−/− and Des+/+ mice.
When pressure was set at 75 mm Hg, myogenic tone developed (31 μm active tone or 21±4% of passive diameter, n=10) in NF arteries isolated from Des+/+ mice. Myogenic tone was not different in HF arteries (22±4%, n=10), and it was lower in LF arteries (15±3%, n=10). In Des−/− mice, myogenic tone was similar to that in Des+/+ mice (not shown). Stepwise increases in flow in isolated arteries induced a significant dilation (Figure 3). Flow-mediated dilation was significantly higher in HF arteries than in NF arteries in Des−/− and Des+/+ mice (Figure 3). Conversely, flow-mediated dilation (FMD) was significantly lower in LF arteries than in NF arteries in Des−/− and Des+/+ mice (maximal dilation was 3.4±1.2 μm in Des−/− mice and 4.0±1.1 μm in Des+/+ mice, n=10 per group).
Inhibition of NO synthesis (L-NAME) significantly attenuated FMD (Figure 3). This inhibition was more pronounced in HF than in NF arteries (94±6% versus 67±10% inhibition, respectively; n=10 per group). The absence of desmin did not change the effect of L-NAME (93±8% versus 67±12% inhibition in HF versus NF arteries, respectively; n=10 per group).
The expression of eNOS was significantly higher in HF arteries than in NF arteries (Figure 3, bottom). eNOS expression was not different in LF arteries versus NF arteries (Figure 3). No significant difference in eNOS expression was found between Des+/+ and Des−/− mice (Figure 3). In the same arteries, a Western blot analysis of actin was performed. No change in actin expression was found in HF and LF arteries, relative to NF arteries, and the absence of desmin had no effect on the expression of actin (data not shown).
Dilation induced by the NO donor SNP (see online Figure II, top, available at http://atvb.ahajournals.org) was not affected by the increase in blood flow in either mouse strain. On the other hand, in LF arteries, SNP-induced dilation was strongly attenuated in both strains. SNP-induced dilation was significantly lower in Des−/− than in Des+/+ mice.
Phenylephrine (online Figure II, middle) and calcium (online Figure II, bottom) induced a concentration-dependent contraction of mesenteric resistance arteries. Phenylephrine- and calcium-induced contractions were significantly lower in arteries from Des−/− mice compared with Des+/+ mice. In Des+/+ mice, phenylephrine- and calcium-induced contractions were not affected by the increase in blood flow, but they were markedly decreased after a reduction in blood flow. In Des−/− mice, phenylephrine- and calcium-induced contractions were significantly decreased in HF arteries, and this reduction was more pronounced in LF arteries.
This is the first study showing a relationship between the absence of desmin and an exaggerated structural adaptation (remodeling) in response to changes in blood flow.
The model used allows the study of resistance arteries from the same arterial bed subjected to different levels of blood flow under the same conditions of pressure and circulating environment. This model had been initially described in rats.13,14⇓ In previous studies, increases and decreases in flow have been reported to respectively induce outward and inward arterial remodeling.10,13,14,22,23⇓⇓⇓⇓ This structural adaptation allows normalization of wall shear stress and is accompanied by a compensatory change in medial mass to restore circumferential wall tensile stress. Changes in arterial smooth muscle cell size and number also participate in this arterial remodeling in response to altered blood flow.11,13,14,22⇓⇓⇓
In the present study, the response of mouse resistance arteries to changes in flow was comparable to that previously found in rat small arteries11,13,14,22⇓⇓⇓ and large arteries.9,10⇓ In control (Des+/+) mice, the increase in flow induced a rise in arterial diameter, as previously shown in large arteries9,10⇓ and small arteries.11,13,14,22⇓⇓⇓ In LF arteries, the remodeling was rapid, and in agreement with previous studies performed in rats,13,14⇓ the reduction in blood flow resulted in inward remodeling accompanied by an hyporeactivity of smooth muscle and endothelium.
Flow-mediated vascular remodeling depends mainly on the capacity of the endothelium to produce vasodilator agents. Inhibition of NO synthesis strongly impairs flow-induced remodeling in large arteries.9 In small arteries, this issue remains controversial because chronic NO synthesis blockade does not prevent flow-induced remodeling in rat mesenteric arteries in a model similar to the one used in the present study.24 Nevertheless, we found increased eNOS expression in HF arteries, in agreement with a previous study performed under similar conditions, in the rat.11 In addition, pharmacological treatments that improve FMD, such as ACE inhibitors, improve angiogenesis, suggesting a beneficial effect of FMD on vascular remodeling.25 Thus, our results support, although they do not demonstrate, the concept that flow, by stimulating the expression of eNOS and consequently the production of NO, might play a role in vascular wall remodeling. Nevertheless, another pathway(s) might be stimulated in parallel.24
In mice lacking desmin, we found significantly lower contractility of vascular smooth muscle, in agreement with our previous report.4 Also in agreement with our previous study, we found that flow- and SNP-induced dilations were decreased in Des−/− mice, suggesting that the defect is located at the level of the smooth muscle. These observations, combined with the present finding that eNOS expression in Des−/− mice was identical to that in Des+/+ mice, confirm that endothelial function is not affected by the absence of desmin.
In Des−/− mice, mesenteric arteries overadapted in response to changes in blood flow. A possible explanation could be that the low muscular tone found in Des−/− mice causes an exaggerated structural response to flow. Indeed, one may consider the vascular response to a flow stimulus as a balance between endothelial stimulation by flow, which activates the remodeling process, and the smooth muscle–dependent contractile tone, which counteracts the effect of FMD. Indeed, resistance arteries possess a basal contractile tone26,27⇓ that is continuously counteracted by FMD. Thus, the low contractility that was found in Des−/− mice arteries4 could counteract flow-induced remodeling to a lesser extent than in control mice. In addition, we found that the increase in flow enhanced NO production by the endothelium and eNOS expression. This was identical in Des+/+ and Des−/− mice, consistent with the assumption that endothelial function was normal in Des−/− mice. Nevertheless, this issue would require further confirmation. Other explanations are possible. The absence of a key structural cytoskeletal protein such as desmin might change the mechanical behavior of cells, not in the short term (no change in pressure-diameter relationship was observed in Des−/− mice) but in the long term. This latter explanation is supported by our previous finding that vimentin-null mice also show an excessive diameter enlargement when submitted to a chronic increase in flow in the carotid artery.23 Thus, the absence of 1 of the 2 main intermediate filaments, desmin or vimentin, leads to the same effect (an excessive arterial enlargement in response to a chronic change in blood flow). In addition, flow-mediated endothelium-dependent dilation is lower in vimentin-null mice than in control mice.16 This observation and the low contractility found in Des−/− mice4 suggest that the changes in acute vascular reactivity are not sufficient to determine the structural adaptation in response to long-term changes in the hemodynamic environment. The overadaptation of mesenteric resistance arteries to changes in hemodynamic conditions might reflect a higher blood flow need in downstream tissues. Indeed, the absence of desmin has been associated with a decreased mitochondrial function and energy supply,5 which might be the cause of lower calcium handling,6 potassium channel activity,7 and phospholipase activity.8 Indeed, the cardiomyopathies described in mice lacking desmin2,3⇓ could be associated with this low mitochondrial function. In addition, the exaggerated enlargement in HF arteries was associated with an exaggerated diameter reduction in LF arteries. This might be deleterious in ischemic tissues, in which arteries located downstream from an occlusion and subjected to LF would excessively reduce their diameter.
In conclusion, changes in blood flow in mice lacking desmin induced an exaggerated structural adaptation of resistance arteries. This might be due to an imbalanced flow-dependent remodeling or to a higher blood flow need in tissues fed by resistance arteries. This overadaptation might also be a consequence of a defect in cytoskeleton capacity to rearrange after stimulation.
This study was supported in part by a grant from the French Association Against Myopathies (Association France-Myopathies), Paris, France. Dr Loufrani was a fellow of the French Foundation for Medical Research, Paris, France.
Received April 12, 2002; revision accepted June 3, 2002.
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- ↵Li Z, Mericskay M, Agbulut O, Butler-Browne G, Carlsson L, Thornell LE, Babinet C, Paulin D. Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle. J Cell Biol. 1997; 139: 129–144.
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- ↵Tronc F, Wassef M, Esposito B, Henrion D, Glagov S, Tedgui A. Role of NO in flow-induced remodeling of the rabbit common carotid artery. Arterioscler Thromb Vasc Biol. 1996; 16: 1256–1262.
- ↵Tuttle JL, Nachreiner RD, Bhuller AS, Condict KW, Connors BA, Herring BP, Dalsing MC, Unthank JL. Shear level influences resistance artery remodeling: wall dimensions, cell density, and eNOS expression. Am J Physiol. 2001; 281: H1380–H1389.
- ↵Koller A, Huang A, Sun D, Kaley G. Exercise training augments flow-dependent dilation in rat skeletal muscle arterioles: role of endothelial nitric oxide and prostaglandins. Circ Res. 1995; 76: 544–550.
- ↵Unthank JL, Fath SW, Burkhart HM, Miller SC, Dalsing MC. Wall remodeling during luminal expansion of mesenteric arterial collaterals in the rat. Circ Res. 1996; 79: 1015–1023.
- ↵Loufrani L, Matrougui K, Gorny D, Duriez M, Blanc I, Levy BI, Henrion D. Flow (shear stress)-induced endothelium-dependent dilation is altered in mice lacking the gene encoding for dystrophin. Circulation. 2001; 103: 864–870.
- ↵Matrougui K, Loufrani L, Heymes C, Levy BI, Henrion D. Activation of AT(2) receptors by endogenous angiotensin II is involved in flow-induced dilation in rat resistance arteries. Hypertension. 1999; 34: 659–665.
- ↵Loufrani L, Lehoux S, Tedgui A, Levy BI, Henrion D. Stretch induces mitogen-activated protein kinase activation and myogenic tone through 2 distinct pathways. Arterioscler Thromb Vasc Biol. 1999; 19: 2878–2883.
- ↵Matrougui K, Tanko LB, Loufrani L, Gorny D, Levy BI, Tedgui A, Henrion D. Involvement of Rho-kinase and the actin filament network in angiotensin II-induced contraction and extracellular signal-regulated kinase activity in intact rat mesenteric resistance arteries. Arterioscler Thromb Vasc Biol. 2001; 21: 1288–1293.
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- ↵Schiffers PM, Henrion D, Boulanger CM, Colucci-Guyon E, Langa-Vuves F, van Essen H, Fazzi GE, Levy BI, De Mey JG. Altered flow-induced arterial remodeling in vimentin-deficient mice. Arterioscler Thromb Vasc Biol. 2000; 20: 611–616.
- ↵Ceiler DL, De Mey JG. Chronic NG-nitro-l-arginine methyl ester treatment does not prevent flow-induced remodeling in mesenteric feed arteries and arcading arterioles. Arterioscler Thromb Vasc Biol. 2000; 20: 2057–2063.
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