Published online before print February 21, 2008, doi: 10.1161/ATVBAHA.107.153056
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© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Sequential Activation of Matrix Metalloproteinase 9 and Transforming Growth Factor β in Arterial Elastocalcinosis
From the Faculty of Pharmacy (C.B., S.M., J.B., P.M.) and the Department of Pharmacology, Faculty of Medicine (D.d.B.), University of Montreal, Québec, Canada.
Correspondence to Pierre Moreau, PhD, Professor and Dean, Faculty of Pharmacy, Université de Montréal, 2940 Chemin de la polytechnique, Room 2143, P.O. Box 6128, Station "Centre-Ville", Montréal, Québec, H3C 3J7, Canada. E-mail pierre.moreau{at}umontreal.ca
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Abstract |
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Objective— Isolated systolic hypertension is associated with increased elastase activity, vascular calcification, and vascular stiffness. We sought to determine the importance of elastase activity and matrix degradation in the development of elastocalcinosis.
Methods and Results— Elastocalcinosis was induced in vivo and ex vivo using warfarin. Hemodynamic parameters, calcium deposition, elastin degradation, transforming growth factor (TGF)-β signaling, and elastase activity were evaluated at different time points in the in vivo model. Metalloproteinases, serine proteases, and cysteine proteases were blocked to measure their relative implication in elastin degradation. Gradual elastocalcinosis was obtained, and paralleled the elastin degradation pattern. Matrix metalloproteinase (MMP)-9 activity was increased at 5 days of warfarin treatment, whereas TGF-β signaling was increased at 7 days. Calcification was significantly elevated after 21 days. Blocking metalloproteinases activation with doxycycline and TGF-β signaling with SB-431542 were able to prevent calcification.
Conclusions— Early MMP-9 activation precedes the increase of TGF-β signaling, and overt vascular elastocalcinosis and stiffness. Modulation of matrix degradation could represent a novel therapeutic avenue to prevent the gradual age-related stiffening of large arteries, leading to isolated systolic hypertension.
The objective was to determine the importance and timing of matrix degradation in relation to elastocalcinosis associated with enhanced vascular stiffness. Matrix degradation and its cellular signaling are essential to elastocalcinosis and precede elastin fragmentation, overt calcification, and enhanced vascular stiffness. These early events could represent means to limit stiffening of large arteries.
Key Words: vascular calcification • elastocalcinosis • elastases • extracellular matrix • MMP-9 • TGF-β
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Introduction |
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With aging, alterations of the vascular structure and function occur, including medial arterial calcification (medial elastocalcinosis MEC) or elastocalcinosis and elastin degradation.1 These modifications induce gradual stiffening of arteries which contributes to the development of isolated systolic hypertension (ISH), the predominant type of hypertension in elderly patients.
Elastocalcinosis is characterized by a deposition of hydroxyapatite on the elastic lamellae of arteries. It occurs independently of atherosclerosis.2 Until recently, it was considered as a passive process, taking place with time. However, several studies demonstrated that vascular calcification is an active phenomenon controlled by serum and matrix proteins, such as matrix Gla protein.3 Moreover, it involves phenotypic changes of vascular smooth muscle cells with the expression of bone-related proteins.4
Aging is associated with an increased collagen/elastin ratio explained in part by an enhanced degradation of elastin.5,6 Indeed, elastase activity, mainly endopeptidases, including cysteine proteases, serine proteases, and metalloproteinases, is increased with age in human aortas.7 In the context of aging and vascular calcification, metalloproteinases, especially matrix metalloproteinases (MMPs), have been thoroughly studied. Investigations on the genetic mutations of MMPs demonstrated its contribution to age-related large artery stiffening.8,9 Furthermore, enhanced MMP-9 and MMP-2 levels and serum elastase activity were observed in patients with ISH and correlated independently with PWV.10 In 2000, Vyavahare et al showed for the first time that elastin calcification was blunted by a site specific delivery of MMP inhibitor.11 Moreover, Qin et al demonstrated, in an animal model, that matrix metalloproteinase inhibition with doxycycline and GM6001 decreased hydroxyapatite accumulation in the aorta.12 These results suggest that MMPs and elastin degradation are involved in MEC.
Elastin degradation induces the release of soluble elastin peptides and TGF-β1.13 These peptides interact with elastin-laminin receptor (ELR)14 and TGF-β receptor, respectively. Simionescu et al demonstrated that elastin peptides (through ELR) and TGF-β1 induced phenotypic changes of VSMCs with osteogenic responses.15 Moreover, TGF-β stimulated vascular cells with an osteogenic phenotype to calcify.16
Therefore, evidence is mounting that MMPs and elastin degradation are implicated in the calcification process. However, many questions remain unanswered. Indeed, we do not know the implication of MMPs in a physiopathological model that seems relevant to the human condition. Moreover, when the enzymes intervene in the process is also an open question. Finally, do MMPs act alone or do other elastases also contribute? This study aimed at answering these uncertainties. Our working hypothesis is that elastase activity and extracellular matrix degradation are essential to the early development of arterial elastocalcinosis in ISH and that MMPs are not the only elastases implicated.
To test this hypothesis, we used an animal model of arterial elastocalcinosis based on an impairment of matrix Gla protein (MGP) carboxylation, a protein able to prevent calcification. Recently, undercarboxylated MGP was associated with vascular calcification in patients.17 High doses of warfarin were administrated to prevent the vitamin K–dependent MGP carboxylation, whereas bleeding was prevented by concomitant administration of phylloquinone (vegetal vitamin K quinone) used by the liver but not by the vessels for protein carboxylation. With this model, we previously observed a specific accumulation of hydroxyapatite on elastin fibers.18,19 We also demonstrated an increased collagen/elastin ratio18,20 and phenotypic changes in vascular wall demonstrated by changes in osteopontin expression.21
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Methods |
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In Vivo Experiments
Treatments
Male Wistar rats (initial weight of 175 to 200 g, n=6 to 20 per group) were obtained from Charles River Breeding Laboratories (St Constant, Qc, Canada). They received warfarin (20 mg . kg–1 . day–1 in drinking water) and vitamin K (phylloquinone, 15 mg . kg–1 . day–1 subcutaneous injection) (WVK) during 1, 3, 5, 7, 14, 21, and 28 days. Dosages were adjusted every second day. Controls consisted of age-matched untreated rats (Ctrl). In additional rats, doxycycline, a nonselective MMP inhibitor, was administrated (30 mg . kg–1 . day–1, in the food) during 14 days, from day 1 to day 14 (WVK28+Doxy1 to 14) or day 15 to day 28 (WVK28+Doxy15 to 28) and during 28 days (WVK28+Doxy1 to 28), concomitantly with 28 days of WVK. All animal experiments were approved by the Animal Care and Use Committee of Université de Montréal.
Hemodynamic Parameters
Animals were anesthetized with pentobarbital (65 mg/kg), and short catheters (polyethylene-50, approx. 10 cm, Folioplast SA) were inserted into the distal abdominal aorta through the left femoral artery and into the aortic arch through the left carotid. Catheters were connected to a pressure transducer to allow the measurement of systolic (SBP) and diastolic blood pressures (DBP) at each location. Mean arterial blood pressure (MBP) as well as carotid and femoral pulse pressures (PP) were calculated from these parameters. Pulse wave velocity (PWV) was measured by the foot-to-foot method from the 2 signals, as previously described.18 Finally, the aorta was harvested. Portions were frozen at –80°C for calcium amount, Western blot analysis, and elastases activity evaluation. A section of aorta was fixed in 4% cacodylate-buffered paraformaldehyde and embedded in paraffin blocks for histological evaluation.
Vascular Wall Composition
To measure calcium content, sections of the aorta were dried at 55°C and calcium was extracted with 10% formic acid (30 µL/mg of dry tissue) overnight at 4°C. The colorimetric quantification was achieved through a reaction with o-cresolphtalein (Teco Diagnostics).
Fragmentation of the medial elastic fiber network (excluding the external and internal laminae) and calcium deposition were evaluated on 7-µm thick sections stained with Weigert solution22 and von Kossa, respectively. The number of elastin breaks was evaluated by a blinded observer.
Elastolytic Activity
Proteins were extracted from aorta using 20 mmol/L Tris HCl pH 7.5, 5 mmol/L EGTA, 150 mmol/L NaCl, 20 mmol/L Glycerophosphate, 10 mmol/L NaF, 1% Triton X-100, 0,1% Tween 20. Elastase activity was evaluated using EnzChek elastase activity assay kit (Molecular Probes by Invitrogen). This kit contains DQ elastin that becomes fluorescent once digested by elastases and proteases. After incubation at 37°C during 18 hours, the fluorimetric reaction was revealed at 505/515 nm. The results were normalized with porcine pancreatic elastase and expressed as elastolytic activity of pancreatic elastase equivalent units. To determine the different families of elastases involved, antiproteases were added to the samples: aprotinin (2 µg/mL) for serine proteases, 1,10 phenanthroline (10 mmol/L) for metalloproteinases, and E-64 (10 µmol/L) for cysteine proteases.
For Western blot, immunoprecipitation, and zymography analysis, proteins were extracted from rat aorta using 20mmol/L Tris HCl pH 7.5, 5 mmol/L EGTA, 150 mmol/L NaCl, 20 mmol/L Glycerophosphate, 10 mmol/L NaF, 1% Triton X-100, 1 mmol/L sodium orthovanadate, 0,1% Tween 20, 1 µg/mL aprotinin, and 1 mmol/L PMSF.23
MMPs and TIMPs Activity
MMPs activity was measured by standard gelatin zymography. Sample, renaturation, and incubation buffers were purchased from Biorad. SDS-polyacrylamide gels were incubated 36 hours at 37°C. Active MMP-2 and MMP-9 were localized at 62 kDa and 82 kDa, respectively, and visualized as areas of lytic activity on an otherwise blue background. TIMPs activity was evaluated by reverse zymography. A mix of human MMP-2/MMP-9 (Chemicon Inc) was incorporated into gelatin-containing zymogram gel at 0.13 µg/mL.24 Active TIMP-1 (27 kDa) and TIMP-2 (21 kDa) were visualized as undigested bands (dark blue) on an otherwise clear background. Activity was expressed in arbitrary units and normalized by control values (presented as 100).
Western Blot
For Western blot analysis, equal amounts of proteins (30 µg) were resolved by electrophoresis on 12% PAGE and transferred to nitrocellulose membranes. Afterward, membranes were incubated overnight at 4°C with Cbfa-1 antibody (Chemicon, 1: 200 in 5% nonfat milk). After 4 washes of 5 minutes with TTBS, membranes were incubated with antirabbit secondary antibody (New England BioLabs) 1: 2000 for 45 minutes at room temperature. Immunoreactive bands were then revealed with ECL reactive (GE Healthcare) using the Typhoon scanner 9410. Protein loading normalization was established using a β-actin antibody 1: 5000 (Sigma), and the results were expressed as percent change relative to controls.
For immunoprecipitation analysis, 350 µg of protein extracts from rat aorta were incubated overnight at 4°C with polyclonal Smad 2/3 antibody (New England BioLabs, 1: 100). The immune complexes were then collected with protein G sepharose beads. Binding of Smad 2/3 to Smad 4 was assayed by Western blot using Smad 4 antibody (New England BioLabs, 1: 1000) corrected by Smad 2/3 protein content.
Ex Vivo Experiments
Aortic segments of untreated Wistar rats were placed into DMEM (Hyclone by Fisher scientific) containing 1.8 mmol/L Ca2+ and 0.9 mmol/L PO43– with penicillin and streptomycin. Medium was maintained at 37°C in a 5% CO2 atmosphere and changed every 2 days. To induce calcification, 10 µmol/L of warfarin was added to the medium. The concentration of PO43– was increased to 3.8 mmol/L, 2 days after the addition of warfarin. The aortic rings were placed in this medium with high PO43– and warfarin during 4 days (CM). Thereafter, they were cleaned in PBS and frozen at –80°C or fixed in paraformaldehyde for paraffin sectionning. For viability staining, the aortic rings were incubated with 0.5 mg/mL methylthiazolyldiphenyl-tetrazolium bromide (MTT) in DMEM for 3 hours at 37°C and washed in physiological saline 3 times, and then embedded for frozen sectioning. Calcification was assessed by both von Kossa staining and colorimetric quantification.
To determine the implication of different families of elastases in vascular calcification, antiproteases were added to the media: aprotinin (2 µg/mL) for serine proteases, E-64 (10 µmol/L) for cysteine proteases, 1,10 phenanthroline (10 mmol/L) or doxycycline (100 µg/mL) for metalloproteinases. Moreover, the addition of SB-431542 (10 µmol/L), an inhibitor of activin receptor-like kinase, and lactose (5 mmol/L), an elastin-laminin receptor antagonist allowed to evaluate the involvement of TGF-β1 and elastin-derived peptides in this process.
Drugs and Statistical Analysis
All drugs were purchased from Sigma Chemical Co unless specified otherwise. Values are expressed as mean±SEM. An ANOVA followed by Bonferonni’s correction was used to compare the groups. We used the 1 sample t test and fixed control values at 100 in zymography analysis.
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Results |
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In Vivo Experiments
Hemodynamic Parameters
WVK treatment induced isolated systolic hypertension after 28 days. This was associated with an enhanced pulse pressure, suggesting increased arterial rigidity. In fact, an elevated vascular stiffness was observed by measuring the pulse wave velocity, a blood pressure independent parameter. From this method, gradual stiffening (PWV) of the aorta was observed with WVK treatment, reaching significance after 21 days (Figure 1A), whereas the maximum value was obtained after 28 days of treatment.
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Vascular Wall Composition
Calcium content in the aorta followed a similar pattern as the one observed for PWV. Indeed, a gradual increase of vascular calcification was observed from day 7 to day 28 of WVK treatment. This increase was significant after 21 days of WVK treatment. However, the maximum value was not obtained until the 4th week of treatment (Figure 1A). This calcium deposition was localized on the elastic lamellae (Figure 1B).
The breaks in the elastic network of the vascular wall perfectly matched the kinetic defined by vascular calcification. As demonstrated in the Table, this parameter was also significantly increased compared to controls after 21 days of WVK treatment, and reached its maximum after 28 days.
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Phenotypic Changes
In line with the increase in vascular calcification, we observed a time-dependant increase in the expression of Cbfa-1, evaluated by Western blot normalized with β-actin (n=5 per group). The increase reached significance after 21 days of WVK treatment (WVK 21days: 0.300±0.035 versus Ctrl: 0.122±0.017, P<0.05) and plateaud at 28 days (0.315±0.053, P<0.05; see supplemental data, Figure A, available online at http://atvb.ahajournals.org).
Ex Vivo Model of Elastocalcinosis
To assess cell viability, aortic rings were incubated with MTT at the end of a 6-day culture period. The smooth muscle cells in the media of the vessels exposed to the calcifying media were stained. This did not differ from fresh aortas. Moreover, aortic rings exposed to a noncalcifying media presented a similar staining (data not shown). Incubation of aortic sections in DMEM with warfarin and high concentration of PO43– induced a significant increase of calcium amount in the vascular wall (Ctrl: 0.35±0.05 versus CM: 4.41±1.08 µg/mg of dry tissue, P<0.05). Von Kossa staining showed calcification on the elastin lamellae of aortas incubated in calcifying media (Figure 1C).
Elastolytic Activity In Vivo
Elastolytic activity was already increased after 7 days of WVK treatment, and the augmentation was significant and maximal after 14 days of WVK treatment (Figure 2A). At this time point, 1,10-phenanthroline, the inhibitor of metalloproteinases, completely suppressed elastolytic activity (Figure 2B). However, serine and cysteine proteases inhibitors did not decrease significantly elastase activity at 14 days. 1,10-phenanthroline reduced elastolytic activity at all time-points (data not shown).
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P<0.05 vs CM.
Elastases Implication Ex Vivo
In this direction, inhibition of different families of elastases demonstrated that only metalloproteinases seemed to be important in calcification, using our ex vivo model. Indeed, 1,10-phenanthroline and doxycycline, 2 nonselective inhibitors of metalloproteinases, significantly prevented calcification, whereas aprotinin and E-64 did not (Figure 2C).
MMPs and TIMPs Activity In Vivo
Gelatin zymography on aortic extracts allowed visualization of MMP-9 and MMP-2 activity. Whereas MMP-2 activity remained unchanged during WVK treament, a transient and significant increase of MMP-9 activity was observed after 5 days of WVK treatment (Figure 3A). MMP-9 activity went back to a normal level at 14 days of WVK treatment. These variations of MMP activity were not associated with changes of TIMP-1 or TIMP-2 activity (data not shown).
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The implication of metalloproteinases in the elastocalcinosis process was confirmed by doxycycline treatment in the rat model. When doxycycline was added to the WVK treatment, both calcium content (Figure 3B) and elastin fragmentation (Table) were significantly prevented with the 3 administrations regimen of doxycycline. However, only 28 days of doxycycline could successfully prevent the increase in vascular stiffness (Table). Indeed, administration of doxycycline for the first or second half of the WVK treatment had no effect on PWV. Moreover, 28 days of doxycycline partially reduced Cbfa-1 expression (45±22%, ns).
TGF-β Signaling
TGF-β signaling, evaluated by smad 2/3–smad 4 binding, increased in a rapid and transitory fashion. It reached significance as early as 7 days of WVK treatment and then returned to control levels (Figure 4A).
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The implication of some products of ECM degradation was evaluated with the use of lactose and SB-431542 (Figure 4B) ex vivo. This inhibitor of the kinase activity of TGF-β receptor prevented the increase of calcium deposition in our ex vivo model. However, lactose, an antagonist of elastin-derived peptides receptor, did not prevent aortic calcification, although it reduced extracellular signal regulated kinase (ERK) 1/2 phosphorylation, a downstream effector (supplemental Figure B). The association of lactose with SB-431542 produced a reduction of calcium amount similar to the one induced by SB-431542 alone.
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Discussion |
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Herein, we establish for the first time a sequence of extracellular matrix events taking place in the development of elastocalcinosis and vascular stiffness. Our results suggest an essential implication of early MMP-9 and TGF-β signaling, in the process.
WVK treatment in vivo increased systolic blood pressure (SBP) without diastolic blood pressure (DBP) change, thus increasing pulse pressure (PP). This was associated with increased calcification and elastin degradation, which were accompanied by increased PWV. Because these changes occur in humans with ISH,18,19 these data lend support to the validity of our model. However, a surprising observation was the transitory fall in PWV in WVK7 rats compared to controls. This could be explained by the early activation of elastases, such as MMP-9, which could trigger matrix degradation before calcification, thus "loosening" the vascular walls. Interestingly, elastic arteries show 2 physical changes during aging. Before stiffening, arteries dilate. O’Rourke et al suggested that this dilation could be a consequence of fracture of elastic lamellae. Through a transfer of stresses to collagen, this fracture could participate also to arterial stiffening.25
Elastin presents a high affinity for calcium.26 Seligman et al demonstrated that elastin mineralization in vitro increased with the age of the individual from whom the vessel was obtained.27 This was associated with an augmentation of polar amino acids in the elastin samples form older patients.27–29 Moreover, mineralized elastin was associated with a higher content of N-terminal amino acids, indicative of its degradation.6 Therefore, elastin degradation seems to favor calcification through an increase of polarity, which enhances elastin affinity for calcium. The close association between elastin breaks and calcification observed in the present study lends support to this concept.
The degradation of elastin requires elastase activation. In our in vivo model of elastocalcinosis, we observed an increase after 2 weeks of WVK treatment. This elastase activity was persistently reduced by 1,10 phenanthroline, suggesting the implication of metalloproteinases, as measured by elastin DQ degradation. Among metalloproteinases, the metzincins and especially the matrix metalloproteinases (MMPs) are the most studied. The involvement of MMPs in vascular calcification and elastin degradation has already been studied. Indeed, Bailey et al showed that pretreating elastin with AlCl3 not only prevented its degradation by decreasing the activity of MMPs, but also reduced its subsequent calcification.30 Furthermore, Basalyga demonstrated, in an arterial injury model involving the periarterial application of calcium chloride (CaCl2), that MMP knock-out mice were resistant to elastin degradation and calcification.31 Thus, evidence is mounting to confirm the role of MMPs in the calcification process. Herein, we demonstrated that WVK treatment increased selectively MMP-9 activity in an acute and transient fashion, after only 5 days. The transient increase of MMP-9 activity cannot explain the significant augmentation of elastase activity after 14 days. Because no change in MMP-2 activity was observed, other MMPs or metalloproteinases could be involved in this later phase, as suggested by the elastase activity assay. Indeed, these results ruled out significant implication of cysteine and serine proteases in the process of elastocalcinosis, and were confirmed with the ex vivo model of elastocalcinosis. In this ex vivo model, aprotinin was used to block serine proteases, E-64 to inhibit cysteine proteases, and 1,10-phenanthroline and doxycycline to reduce metalloproteinases activity. Of the inhibitors examined, only the metalloproteinase inhibitors were able to partially prevent calcification, thus strengthening their implication in the process. Although the nature of the metalloproteinase involved later in the process needs to be resolved, we also have to rule out a potential direct binding of metalloproteinases inhibitors to calcium cations.
Recently, the implication of MMPs in calcification was also demonstrated in a vitamin D3 model, where their inhibition with either doxycycline or GM6001 prevented vascular calcification.12 Several mechanisms have been proposed for the actions of doxycycline, a tetracycline, on the extracellular matrix: direct inhibition of the enzyme, downregulation of gene transcription, and alteration of enzyme processing associated with pro-MMP activation.32,33 In our experiments, doxycycline prevented the development of calcification and elastin degradation when used during 2 weeks or during the whole duration of WVK treatment, confirming the central role of MMPs. However, only 28 days of doxycycline prevented the PWV increase, suggesting that not only elastin degradation and calcification are implicated in arterial stiffness, but also fibrosis could intervene in this process.34
As mentioned earlier, degraded elastin presents an increased affinity for calcium. However, extracellular matrix degradation by elastases also induces the release of soluble elastin peptides and TGF-β13 that can influence the process of elastocalcinosis. Soluble elastin peptides present biological effects mediated by the ELR, leading to phenotypic changes characterized by the synthesis of bone proteins like type I collagen35 and Cbfa-1.15 TGF-β is a potent multifunctional regulator of cell growth and differentiation. TGF-β1, a member of the TGF-β superfamily, induces different cellular responses, including stimulation of osteogenesis.36 After TGF-β binding to its receptor, the associated kinase, target of SB-431542, is activated and phosphorylates Smads. Once phosphorylated, Smad-2/3 interacts with co-Smad or Smad-4. This complex moves into the nucleus to stimulate the transcription of several target genes.37 Smads interact with Runx2/Cbfa1, a transcription factor required in osteoblast gene expression, to increase its transactivation ability.38,39 In the present experiments, a transient increase of Smad2/3 to 4 complex formation was observed early during the process of elastocalcinosis (after 7 days of WVK treatment), suggesting an enhanced stimulation of target genes, like Cbfa1. Although the present study does not show an increased transcriptional activity of Cbfa-1, we observed a significant increase of its expression after 3 weeks of WVK treatment, revealing a phenotypic modification in the vascular wall. Therefore, our results support a role for TGF-β in elastocalcinosis as suggested by Simionescu et al.15 Indeed, experiments in the ex vivo model demonstrated that TGF-β inhibition prevented vascular calcification. It is suggested that a reduction in MGP carboxylation could lead to bone morphogenic protein (BMP)-2 release, that can also signal through Smads.40 However, immunohistochemistry was negative for BMP-2 in our model (data not shown) and the activation of Smads was transient, whereas MGP undercarboxylation was sustained.
In contrast to evidence presented by Simionescu, the elastin laminin receptor was not crucial for vascular calcification in our model. Indeed lactose, an ELR antagonist, had no effect on aortic elastocalcinosis, although it blunted downstream signaling of the ELR. It is important to note that our ex vivo aortas maintained viability in culture, as determined with MTT, and that Von Kossa staining revealed the same calcification localization as our in vivo model experiments.
In summary, during the process of vascular medial elastocalcinosis, we observed acute MMP-9 activation, closely followed by the activation of TGF-β signaling. In fact, MMP-9 (activated at day 5) could release TGF-β from the extracellular matrix, inducing the activation of TGF-β signaling observed at day 7. At day 14, an increase of elastase activity by metalloproteinases could be responsible for gradual elastin degradation, augmented at day 14 and significant at day 21. Ex vivo experiments confirmed the crucial role played by MMP-9 and TGF-β signaling. The other elastases evaluated (cystein and serine proteases) were not implicated in elastocalcinosis nor elastin degradation. Through outside-in signaling, increased TGF-β and metalloproteinases could be associated with an increased expression of other proteins, such as endothelin. Previous studies demonstrated a role for endothelin and an increased expression of endothelin in calcified areas is observed at 14 days (Dao HH, Moreau P 2003. Endothelin could be in part responsible for the phenotypic change of VSMCs (Cbfa-1 expression), associated with progressive increases of medial calcification and elastin fragmentation. Thus, this study brings to light a sequence of events leading, through several pathophysiological processes, to enhanced vascular stiffness and isolated systolic hypertension. Continuous research efforts are required to unravel the pathological process leading to large artery stiffness, which is a crucial step to allow the development of new therapies targeted at the arterial wall.
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Acknowledgments |
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We acknowledge the skilled technical assistance of Louise Ida Grondin.
Sources of Funding
This study was supported by the Canadian Institutes for Health Research (CIHR) and the Heart and Stroke Foundation of Canada (Quebec chapter). C.B. received a studentship from the Rx&D/CIHR and S.M. from the Quebec Society of Hypertension. P.M. and D.d.B. are scholars from the Fonds de la Recherche en Santé du Québec.
Disclosures
None.
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Footnotes |
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C.B. and S.M. contributed equally to this study.
Original received September 4, 2007; final version accepted February 9, 2008.
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