Angiotensin II-induced MMP-2 release from endothelial cells is mediated by TNF-{alpha}

Ivan A. Arenas,1,2 Yi Xu,1,2 Patricio Lopez-Jaramillo,3,4 and Sandra T. Davidge1,2

1Department of Obstetrics and Gynecology and 2Department of Physiology, Perinatal Research Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2; and 3Fundacion Cardiovascular del Oriente and 4Universidad Industrial de Santander, Bucaramanga, Colombia

Submitted 17 September 2003 ; accepted in final form 25 November 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiotensin II (ANG II) has been etiologically linked to vascular disease; however, its role in the alterations of endothelial function that occur in vascular disorders is not completely understood. Matrix metalloproteinases (MMPs) and proinflammatory cytokines are involved in the pathological remodeling of blood vessels that occurs in vascular disease. In this study we evaluated the effects of ANG II on tumor necrosis factor (TNF)-{alpha} and MMP-2 production in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with ANG II (0.1–10 µM) for 24 h, in the presence or absence of antagonists of ANG II type 1 (AT1R) and type 2 (AT2R) receptors, and the production and release of TNF-{alpha} and MMP-2 were assessed. ANG II increased TNF-{alpha} mRNA and protein expression and the release of bioactive TNF-{alpha}. Moreover, ANG II induced MMP-2 release and reduced the secretion of tissue inhibitor of MMP (TIMP)-2 from endothelial cells. To elucidate whether endogenous TNF-{alpha} could mediate the effects of ANG II on MMP-2 release, cells were pretreated with anti-TNF-{alpha} neutralizing antibodies or pentoxifylline (an inhibitor of TNF-{alpha} synthesis). TNF-{alpha} inhibition prevented the secretion of MMP-2 induced by ANG II. Furthermore, AT1R antagonism with candesartan prevented the formation of MMP-2 and TNF-{alpha} and the reduction of TIMP-2 induced by ANG II. These results indicate that ANG II, via AT1R, modulates the secretion of TNF-{alpha} and MMP-2 from endothelial cells and that TNF-{alpha} mediates the effects of ANG II on MMP-2 release.

remodeling; vasoactive mediators; inflammation


ANGIOTENSIN II (ANG II) is an important modulator of vascular homeostasis and an important link in the pathophysiology of cardiovascular disease (5, 27). Elevated ANG II and/or increased sensitivity to ANG II have been etiologically associated with major vascular diseases (5, 27). However, most of the studies concerning the effects of ANG II on vascular cells have been conducted in smooth muscle cells. An emerging role for ANG II is through modulation of endothelial cell function. Endothelial cells are essential to maintain normal vascular tone and blood fluidity and to limit vascular inflammation (29). Indeed, a common feature of vascular disorders is the presence of endothelial dysfunction.

Tumor necrosis factor (TNF)-{alpha} has been proposed to be an important mediator of the endothelial alterations seen in vascular disease (10, 21). TNF-{alpha}-stimulated endothelial cells undergo functional alterations resulting in a prothrombotic and proinflammatory phenotype (activation) (20). TNF-{alpha} levels are elevated in a number of vascular disorders, and it appears to be involved in the chronic development of atherosclerosis as well as in the acute plaque events that can result in clinical events such as myocardial infarction or stroke (3, 30). TNF-{alpha} is principally derived from mononuclear phagocytes, but it can also be synthesized in vascular cells such as smooth muscle and endothelium (20). The role of TNF-{alpha} produced in vascular cells is not very well understood; however, it is likely to modulate key vascular processes such as angiogenesis and inflammation (10, 20, 21). Importantly, endothelium-derived TNF-{alpha} could contribute to the pathogenesis of vascular disease.

Interactions between ANG II and TNF-{alpha} may play an important role in the modulation of endothelial function. Some studies have suggested that TNF-{alpha} could mediate the vascular effects of ANG II (11). Interestingly, in endothelial cells, some of the effects of TNF-{alpha}, such as increased free radical production, inflammation, and enhanced remodeling, resemble those attributed to ANG II. Moreover, ANG II has been reported to activate proinflammatory transcription factors in endothelial cells known to induce the formation of TNF-{alpha} (6). Furthermore, ANG II has been shown to stimulate the production of TNF-{alpha} on other vascular cells (18). Altogether, these observations suggest that TNF-{alpha} could mediate some of the effects of ANG II on endothelial function.

Proinflammatory cytokines such as TNF-{alpha} have been shown to induce the release of matrix metalloproteinases (MMPs), including MMP-2 (14, 30). MMPs are a group of zinc-dependent endopeptidases that play a key role in matrix turnover. Indeed, increased interstitial matrix remodeling is believed to be involved in the pathogenesis of atherosclerosis and other vascular disorders (17, 24). MMP-2 participates in the break-down of collagen type IV, a major component of subendothelial basement membrane (19, 28). Moreover, we previously reported (7, 8) that MMP-2, through cleavage of endothelium-derived peptides, may also lead to vasoconstriction and inflammation.

Enhanced MMP-2 activity has been shown to occur in vulnerable atherosclerotic plaques (1). Moreover, higher MMP-2 levels have been reported in patients after acute atherosclerotic events and in women with preeclampsia (22). All of these conditions have been associated with both increased effects of ANG II and higher levels of TNF-{alpha}. However, whether ANG II can induce the release of MMP-2 from endothelial cells is unclear. In this study we evaluated the effects of ANG II on TNF and MMP-2 release from endothelial cells. We hypothesized that ANG II could induce the release of MMP-2 from endothelial cells, in part through the formation of TNF-{alpha}.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All procedures were performed in conformance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society.

Reagents. ANG II, PD-123319, and recombinant TNF-{alpha} were purchased from Sigma, whereas candesartan was obtained from Astra Pharma. Pentoxifylline (no. 002323, Hoechst Canada) was donated by A. Rabinovitch's laboratory (University of Alberta, Edmonton, AB, Canada). Anti-human TNF-{alpha} neutralizing antibodies were obtained from ICN Biomedicals, and M199 medium, L-glutamine, and trypsin were purchased from Life Technologies. Fetal bovine serum (FBS) was purchased from GIBCO and endothelial cell growth factor (ECGF) from VWR International.

Cell culture. Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords as previously described (16). Briefly, the cords were cleaned with PBS and then incubated with collagenase A for 15 min at 37°C. Endothelial cells were collected by centrifugation, and the pellet was resuspended in M199 with 20% FBS. Cells were grown on 0.1% gelatin-coated dishes in M199 with ECGF, heparin, and 20% FBS and used at passages 2–4. Before stimulation HUVECs were plated in six-well plates and incubated in serum-free M199 (without phenol red) containing 0.1% BSA and no ECGF. After stimulation with the different agonists, conditioned medium was collected and cellular protein extracts were prepared or total cellular RNA was extracted.

Measurement of MMP-2 release. MMP-2 release from HUVECs was evaluated in conditioned media by gelatin zymography as previously described (9). Briefly, supernatants were collected and protein content was determined by the Bradford method. One-half microgram of protein was loaded per lane and subjected to 7.5% SDS-PAGE copolymerized with gelatin (2.5 mg/ml). After separation the gels were incubated with Triton X-100 (0.1%; 3 times, 20 min) at room temperature. Subsequently, gels were incubated in enzyme assay buffer containing (mM) 25 Tris, 5 CaCl2, 142 NaCl, and 0.5 Na3N at 37°C for 48 h. To reveal zones of degradation gels were stained with Coomassie blue overnight and then placed in a solution containing 20% methanol and 10% acetic acid for 6 h. Pure human MMP-2/MMP-9 zymography standards (Chemicon) were used as a control. Gels were scanned with Fluor Multimager (Bio-Rad). Preliminary experiments showed that MMP-2 activity detected in conditioned medium by zymography correlated with MMP-2 protein content detected by Western immunoblot (data not shown).

Determination of release of tissue inhibitor of MMP-2. To measure the release of TIMP-2, the tissue inhibitor of MMP-2, a commercial immunoassay (R & D Systems) was carried out according to the manufacturer's instructions. This solid-phase ELISA enables measurements of total human TIMP-2. Supernatants were tested in duplicate for each sample. Absorbance at 450 nm was measured by an automated reader (Hewlett-Packard).

Western blot analysis for TNF-{alpha}. TNF-{alpha} protein expression in endothelial cells was determined by Western blot. Briefly, cell lysates from stimulated HUVECs were collected with lysis buffer (25 mM Tris·HCl pH 7.5 with 0.5% Triton X-100) and sonicated for ~5 s. Protein content was determined by Bradford protein assay. Eighty micrograms of protein were loaded into a SDS-PAGE 15% gel and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were then probed with goat polyclonal anti-human TNF-{alpha} antibody (1:500; Santa Cruz Biotechnology). The primary antibody was then detected with a peroxidase-conjugated donkey anti-goat secondary antibody (1:2,000; Santa Cruz Biotechnology). Membranes were scanned with Fluor Multimager.

Measurement of TNF-{alpha} mRNA. Levels of TNF-{alpha} mRNA were analyzed by real-time reverse transcription (RT)-PCR. The mixture of primers and probe specific for human TNF-{alpha} was purchased from Applied Biosystems. Cell lysates from three different experiments were analyzed. One microgram of total RNA was reverse transcribed. The RT protocol was performed according to the manufacturer's instructions (Biosystems). The RT reaction consisted of 1 µg of total RNA, 1x RT buffer, RNase-free water, 2.5 µM random hexamers, 0.4 U/µl of RNase inhibitor, 5.5 mM MgCl2, 10 mM dNTP mix, and 1.25 U/µl of MultiScribe reverse transcriptase. After incubation at 25°C for 10 min and 60 min at 48°C, the RT enzyme was inactivated by heating at 95°C for 5 min.

The PCR mix consisted of 4 or 2 µl of cDNA from the RT reaction, Taqman universal PCR master mix containing AmpliTaq Gold DNA polymerase, AmpErase UNG, dNTPs with dUTP (catalog no. 4304437, Applied Biosystems), the mixture of primers and TaqMan probe for human TNF-{alpha}, and RNase-free water. Experiments were performed in triplicate for each sample. The PCR mix was initially heated to 50°C for 2 min and then at 95°C for 10 min for optimal AmpErase UNG enzyme activity and to activate AmpliTaq Gold DNA polymerase, followed by 40 cycles of 90°C for 30 s, 60°C for 60 s, and 72°C for 30 s. Average numbers of cycles required to achieve threshold fluorescence (Ct) for ANG II (0.1, 1, 10 µM) were compared with control.

Determination of release of bioactive TNF-{alpha}. The presence of bioactive TNF-{alpha} in conditioned medium was evaluated by the L929-8 bioassay as previously described (4). Briefly, L929-8 cells were cultured in the wells of a 96-well flat-bottomed microtiter plate in medium (Iscove's modified Dulbecco's medium; IMDM) containing 10% FBS plus 2 µg/ml actinomycin D for 2 h at 37° in 5% CO2. Fifty microliters of supernatant or recombinant human TNF-{alpha} standards were then added in triplicate to appropriate wells and cultured at 40°C for 20 h. Cell viability was assessed by incubation for 2 h with neutral red dye (0.05% in PBS), which is taken up by vital cells. The supernatant was removed, the adherent cells were washed with PBS, and color was developed with 0.05 M NaH2PO4 in 50% ethanol. The optical density at 570 nm was measured on an automated microplate reader (Hewlett-Packard) as previously described (4). The lower limit of assay sensitivity is ~200 fg/ml pure recombinant TNF-{alpha}. This bioassay also detects TNF-{beta} but is not affected by other known cytokines.

Statistical analysis. Results are expressed as a percentage of control. HUVECs obtained from different umbilical cords were considered as individual experiments. ANOVA, one way or in ranks, was used to evaluate differences among groups, and the Tukey test for post hoc analysis was applied. Values of P < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of ANG II on MMP-2 and TIMP-2 release. To evaluate the effects of ANG II on MMP-2 and TIMP-2 release, HUVECs were stimulated with ANG II (0.1–10 µM) for 24 h. ANG II stimulation resulted in increased MMP-2 release (Fig. 1). Moreover, to determine the role of ANG II type 1 (AT1R) and type 2 (AT2R) receptors, cells were pretreated with antagonists of AT1R (candesartan; 100 µM) or AT2R (PD-123319; 100 µM) 1 h before ANG II stimulation. Pretreatment with candesartan, but not with PD-123319, prevented the secretion of MMP-2 induced by ANG II (Fig. 2). In similar cell culture conditions, ANG II did not affect the secretion of MMP-9 (92 kDa). Furthermore, ANG II reduced the secretion of TIMP-2 from endothelial cells, which was also inhibited by AT1R antagonism (Fig. 3). AT2R antagonism did not affect ANG II-induced TIMP-2 release.



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Fig. 1. Angiotensin II (ANG II) induces endothelial release of matrix metalloproteinase (MMP)-2. A: representative gel showing an increase in MMP-2 (72 kDa) activity after ANG II (AII) stimulation. M, marker. B: summary of densitometric analysis for MMP-2 activity (n = 7). Data are presented as % of control (±SE). *P < 0.05 vs. control.

 


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Fig. 2. ANG II type 1 (AT1R) but not type 2 (AT2R) receptor antagonism prevents MMP-2 release from human umbilical vein endothelial cells (HUVECs) induced by ANG II (10 µM). C, control. A: representative gel showing that pretreatment with the AT1R antagonist candesartan (Cand; 100 µM), but not with PD-123329 (PD; 100 µM), an AT2R antagonist, prevents the ANG II-induced MMP-2 release. B: summary of densitometric analysis for MMP-2 activity (n = 5). Data are presented as % of control (±SE). *P < 0.05 vs. control; #P < 0.05 vs. ANG II alone.

 


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Fig. 3. ANG II reduces endothelial release of tissue inhibitor of MMP (TIMP)-2. Human TIMP-2 was determined by immunoassay in cell culture supernatants after 24 h of stimulation with ANG II (10 µM). The AT1R antagonist candesartan (100 µM), but not PD-123329 (100 µM), an AT2R antagonist, prevented the release of MMP-2 induced by ANG II. Data shown are means ± SE of 3 experiments. *P < 0.05 vs. control; #P < 0.05 vs. ANG II.

 

Effects of ANG II on TNF-{alpha} formation. TNF-{alpha} is first synthesized as an immature peptide (pro-TNF-{alpha}) that is later cleaved by a TNF-{alpha}-converting enzyme (20). After treatment with ANG II (0.1–10 µM) for 24 h, TNF-{alpha} protein was evaluated in cell lysates with an antibody able to detect mature and immature forms of TNF-{alpha}. We found pro-TNF-{alpha} and mature TNF-{alpha} protein levels to be significantly higher in cells stimulated with ANG II compared with control (Fig. 4). The effect of ANG II on TNF-{alpha} mRNA transcription was evaluated by real-time RT-PCR. RNA isolated from control and ANG II-treated cells was reverse transcribed, and first-strand cDNA was further amplified. The average threshold for ANG II-treated cells occurred earlier than in controls [ANG II: 0.1 µM, 32.2 ± 0.1; 1 µM, 30.7 ± 0.3; 10 µM, 30.01 ± 0.1 vs. control: 35.6 ± 0.4 (P < 0.05); Fig. 5], indicating that ANG II induces the formation of TNF-{alpha} mRNA in HUVECs. To elucidate whether the increments in TNF-{alpha} mRNA and protein were translated into release of bioactive TNF-{alpha}, cell culture supernatants from cells treated with and without ANG II were tested with the L929-8 bioassay. We found that ANG II (10 µM) increased the amount of bioactive TNF-{alpha} in supernatants, which was prevented by pretreatment with candesartan (Fig. 6).



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Fig. 4. ANG II enhances endothelial tumor necrosis factor (TNF)-{alpha} protein production. A: representative Western immunoblot showing an increase in the expression of pro-TNF-{alpha} (~26 kDa) and TNF-{alpha} (~17 kDa) after ANG II stimulation. B: summary of densitometric analysis for TNF-{alpha} expression. Data are presented as % of control (±SE; n = 3). *P < 0.05 vs. control (TNF-{alpha}); #P < 0.05 vs. control (pro-TNF-{alpha}).

 


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Fig. 5. ANG II enhances endothelial TNF-{alpha} mRNA production. A: representative real-time RT-PCR, showing real-time signals for different concentrations of ANG II. The amplification was monitored by real-time PCR using green fluorescent dye. Fluorescence intensity is given in arbitrary units. ANG II samples were amplified for 40 cycles (annealing temperature at 60°C). Cell lysates from 3 independent experiments were analyzed. B: summary of the number of cycles required to achieve threshold fluorescence (Ct). Lower Ct values in ANG II-treated cells (0.1 µM: 32.2 ± 0.1; 1 µM: 30.7 ± 0.3; 10 µM: 30.01 ± 0.1 vs. untreated: 35.6 ± 0.4; *P < 0.05 vs. control) indicate an increase in TNF-{alpha} mRNA expression compared with cells treated with medium alone. Experiments were done in triplicate with a negative control as a background.

 


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Fig. 6. ANG II enhances the release of bioactive TNF-{alpha} from endothelial cells. TNF-{alpha} production was quantified with the L929-8 bioassay by comparison to a pure recombinant human TNF-{alpha}. ANG II (10 µM) increased the amount of bioactive TNF-{alpha} in supernatants, which was prevented by pretreatment with candesartan (100 µM). Results represent the means ± SD of triplicate assays. The results of this experiment were similar to those obtained in 2 additional experiments. *P < 0.05 vs. control; #P < 0.05 vs. ANG II alone.

 

Effects of TNF-{alpha} inhibition on ANG II-induced MMP-2 release. To evaluate whether TNF-{alpha} can mediate the effect of ANG II on MMP-2 release, cells were first pretreated with neutralizing antibodies against human TNF-{alpha} 1 h before ANG II stimulation. The concentration of anti-TNF-{alpha} antiserum was calculated based on the ability to neutralize the effects of 50 pg of TNF-{alpha} on L929 cells. TNF-{alpha} blockade significantly reduced (P < 0.05) the secretion of MMP-2 induced by ANG II (Fig. 7). The use of antiserum alone did not affect the basal release of MMP-2. Moreover, pretreatment with pentoxifylline (0.1 and 1 mg/ml), a nonselective phosphodiesterase inhibitor that has been shown to inhibit TNF-{alpha} synthesis, also prevented the release of MMP-2 induced by ANG II (reduction of 40 ± 10% and 110 ± 9%, respectively, compared with ANG II alone; P < 0.05). Pentoxifylline (1 mg/ml) did not significantly change the basal release of MMP-2 from endothelial cells.



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Fig. 7. Antiserum against TNF-{alpha} inhibits endothelial MMP-2 release induced by ANG II (10 µM). A: representative gel showing that pretreatment with anti-TNF-{alpha} antibodies (25 µg/ml) prevented the ANG II-induced MMP-2 release. B: summary of densitometric analysis for MMP-2 activity (n = 5). Data are presented as % of control (±SE). *P < 0.05 vs. control; #P < 0.05 vs. ANG II (10 µM).

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our data indicate that ANG II-induced release of endothelial MMP-2 is mediated by TNF-{alpha}. It has been shown that ANG II may induce a proinflammatory phenotype in endothelial cells (i.e., increase the expression of adhesion molecules) (25). However, to our knowledge, there are no previous studies reporting the effects of ANG II on TNF-{alpha} formation in endothelial cells. The present study shows evidence of TNF-{alpha} generation at the levels of mRNA, protein expression, and function. These observations are clinically relevant because of the key role of ANG II and TNF-{alpha} in the pathogenesis of vascular disorders such as preeclampsia and atherosclerosis and the availability of treatments to antagonize these factors.

In fact, recent studies have shown that TNF-{alpha} antagonism improves endothelial function in patients with chronic heart failure (12) or chronic inflammation (15). Although the concentration of ANG II that results in a maximum effect on TNF-{alpha} production is higher than the reported circulating levels, it is likely that circulating levels underestimate the concentration available in vascular beds. For instance, there is evidence that locally formed (within vascular walls) ANG II may account for part of its vascular effects (2).

The fact that ANG II may induce the formation of active TNF-{alpha} in endothelial cells is intriguing. ANG II induced TNF-{alpha} gene expression as well as the formation of immature and bioactive forms of TNF-{alpha}, which suggests that ANG II stimulates the proteolytic cleavage of pro-TNF-{alpha}. TNF-{alpha} is an inflammatory cytokine proposed to be a mediator of the endothelial alterations seen in cardiovascular disease (10, 20). Indeed, TNF-{alpha} has been shown to induce endothelial dysfunction, inflammation, and apoptosis (10, 20, 21). Interestingly, some of the effects of TNF-{alpha} on endothelial function, such as increased free radical production, inflammation, and enhanced remodeling, resemble those attributed to ANG II. On the other hand, TNF-{alpha} has been shown to protect endothelial cells from apoptosis by inducing platelet-derived growth factor pathways (13). Therefore, endogenous production of TNF-{alpha} could trigger either detrimental or protective pathways to modulate the effects of ANG II on endothelial cells. Understanding the role of interactions between ANG II and TNF-{alpha} on endothelial modulation of vascular function is necessary.

MMP-2 is an important protease involved in normal (angiogenesis or wound repair) and pathological (chronic inflammation and tumor genesis) blood vessel remodeling (19, 28). In this study, we found that ANG II increases the release of MMP-2 from endothelial cells while decreasing the secretion of TIMP-2, the endogenous inhibitor of MMP-2, which was prevented by AT1R antagonism. Therefore, the increased MMP-2 activity after ANG II stimulation could be due to both increased MMP-2 release and decreased TIMP-2 secretion. These observations may indicate that ANG II, via AT1R, could induce matrix turnover by enhancing the release activity of MMP-2.

Interestingly, opposing findings have been reported in vascular smooth muscle cells and fibroblasts, in which ANG II decreased the secretion of MMP-2 (23, 26). These observations suggest a cell-specific effect of ANG II, which is important to understand when using AT1R antagonists clinically. It could be speculated that for smooth muscle cells and fibroblasts ANG II-mediated decrease in MMP-2 release could lead to collagen deposition promoting fibrosis, whereas in endothelial cells ANG II-induced-MMP-2 activity could be involved in other processes such as angiogenesis, thrombosis, and inflammation. Indeed, we have shown (7, 9) that MMP-2, through cleavage of endothelium-derived peptides, can directly promote vasospasm and facilitate leukocyte recruitment.

The present study suggests that a dysfunctional endothelium caused by ANG II may directly affect the development and progression of atherosclerosis and other vascular disorders by enhancing the local production of TNF-{alpha}, a potent inflammatory cytokine postulated to be an important mediator of vascular disease. Although in most vascular disorders upregulation of ANG II, TNF-{alpha}, and MMP-2 have been described, the contribution of TNF-{alpha} and MMP-2 to the pathophysiological effects of ANG II remains to be understood. Furthermore, these findings also suggest that MMP-2 and/or TNF-{alpha} inhibition could have potential therapeutic implications in some vascular disorders in which ANG II plays a role in the pathogenesis of vascular dysfunction.


    ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Sheena Fang for technical assistance and Dr. Larry Guilbert for helpful suggestions.

GRANTS

The Canadian Institute for Health Research supported this study. S. T. Davidge is a Canada Research Chair in Women's Cardiovascular Health and an Alberta Heritage Foundation for Medical Research (AHFMR) Senior Scholar. Ivan A. Arenas is an AHFMR scholarship recipient and a fellow in Tomorrow's Research Cardiovascular Health Professionals (TORCH).


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. T. Davidge, Perinatal Research Centre, 220 HMRC, Univ. of Alberta, Edmonton, AB, Canada T6G 2S2 (E-mail: sandra.davidge{at}ualberta.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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