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© 2007 American Society for Nutrition J. Nutr. 137:1895-1900, August 2007

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Dietary Iron Deficiency Compromises Normal Development of Elastic Fibers in the Aorta and Lungs of Chicks1–3,

Charles H. Hill4, Chris M. Ashwell5, Shelly J. Nolin4, Fred Keeley5, Catherine Billingham5, Aleksander Hinek5 and Barry Starcher6,*

4 Department of Poultry Science, North Carolina State University, Raleigh, NC 27695; 5 Division of Cardiovascular Research and Structural Biology and Biochemistry, Hospital for Sick Children, Toronto, Canada M5G 1X8; and 6 Department of Biochemistry and University of Texas Health Center, University of Texas, Tyler, TX 75708

* To whom correspondence should be addressed. E-mail: barry.starcher{at}uthct.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Elastic fibers play a key role in the structure and function of numerous organs that require elasticity. Elastogenesis is a complex process in which cells first produce a microfibrillar scaffold, composed of numerous structural proteins, upon which tropoelastin assembles to be cross-linked into polymeric elastin. Recently, it was demonstrated that low concentrations of free iron upregulate elastin gene expression in cultured fibroblasts. The present studies were conducted to assess whether low-iron diets would affect the deposition of elastic fibers in an in vivo model. One-day-old chicks were fed semipurified diets containing 1.3 (low), 12 (moderate), and 24 (control) mg/kg of iron. After 3 wk, chicks in the low-iron group were underweight and anemic. Their aortas were smaller with significantly thinner walls than control chicks, yet elastin or collagen content did not decrease relative to total protein. They also demonstrated a significantly lower stress-strain resistance than the controls. Electron microscopy demonstrated that aortic and lung smooth muscle cells were vacuolated and surrounded by loose extracellular matrix and disorganized elastic lamellae with diffuse and fragmented networks of elastic fibers and microfibrils. Immunohistology demonstrated that fibrillin-3 (FBN3) was disorganized and markedly reduced in amount in aortas of the low-iron chicks. Elastin messenger RNA levels were not downregulated in the tissues from the low-iron-fed chicks; however, there was a significant reduction in expression of the FBN1 and FBN3 genes compared with control chicks. The studies indicate that iron deficiency had a pronounced negative effect on elastic fiber development and suggests that fibrillin may have an important role in this pathology.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The aorta is a robust and structured organ made of numerous concentric layers of smooth muscle cells alternating with the extracellular matrix consisting of abundant elastic lamellae and other matrix components, such as collagen fibers, microfibrils, and proteoglycans. Whereas other matrix components provide the aorta with mechanical strength, elastic membranes allow the vessel to expand and contract in response to pulsating blood pressure. Whereas the normal development of aorta is compromised in certain genetic disorders (Marfan Syndrome, Williams-Beuren Syndrome), the effects of extreme metabolic turmoil or a deficiency of essential nutrients during fetal and neonatal life may also contribute to a structural shortfall and future functional failing of this vital organ. Only rarely has a deficiency of an essential nutrient been found to directly affect the aorta. One of the best examples that illustrates the influence of an essential nutrient on development of large vessels came from studies demonstrating that aortas of animals fed copper-deficient diets were mechanically fragile, contained disrupted elastic lamellae, and eventually developed dissecting aneurysms (1–4). This was due to copper's role as a cofactor for lysyl oxidase, the enzyme responsible for the oxidative deamination of epsilon amino groups of lysine and the subsequent formation of the lysine-derived cross-links of elastin and collagen.

The extensive literature regarding the biological effects of iron deficiency contains few studies related to major blood vessels. Those 30-y-old papers reported that iron deficiency resulted in a diminution of mucopolysaccharides and abnormal calcium accumulation in aortic walls of rats fed an iron-deficient diet (5–7). It was also shown that iron deficiency did not prevent development of atherosclerosis in chicks fed a high-cholesterol diet (8) or rats exposed to oral contraceptive steroids (9). Seyama et al. (10) reported that iron deficiency did not affect aortic collagen or cholesterol levels in rats but had a tendency to decrease elastin levels. It has also been reported that abdominal aortas of iron-deficient rats had increased extensibility, resulting in increased arterial diameter; this feature was linked to a flow-dependent remodeling, resulting in degradation of collagen (8). Although iron is required for prolyl hydroxylase activity and the conversion of proline to hydroxyproline (11), there have been no previous reports of collagen defects in iron-deficient animals.

A recent study by Bunda et al. (12) demonstrated that a net deposition of elastin by cultured fibroblasts was modulated by fluctuations of intracellular iron. The authors showed that whereas low levels of iron were needed for activation of the elastin gene, excessive iron levels can downregulate elastin message stability. These data suggest that iron constitutes another important element for the regulation of normal formation of elastic fibers. In this study, we used an in vivo model of neonatal chicks maintained on iron-rich and iron deficient diets. We hypothesized that deprivation of dietary iron could affect normal development of elastic fibers in various organs, such as major blood vessels, lungs, skin, and tendons, but we concentrated on a detailed analysis of aorta and lungs.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Animals and experimental diets. The chicks used in these studies were a white Plymouth Rock cross-bred obtained from the North Carolina Experiment Station farm. They were weighed and started the experiment diets on the day of hatching. The basal diet (Table 1) contained by analysis 1.3 mg/kg iron (low iron). Iron was added as ammonium ferric citrate to supply 12 (moderate) and 24 (control) mg Fe/kg of diet, verified by analysis. Chicks consumed feed and deionized water ad libitum. At 3 wk of age, blood was obtained for hematocrit determination, the chicks were killed by cervical dislocation, and the aortas were removed for biochemical analysis, viscoelasticity measurements, histology, and gene activity assay. The lungs were removed, examined histologically, and assayed for desmosine. Two experiments were conducted with 10–20 chicks in each experimental group. In the gene analysis experiment, 4 chicks from each group of both experiments were used. The values did not differ between experiments and were combined for statistical analysis. All animal experiments conformed to the guidelines for the ethical and humane treatment of animals and all animal research was approved by the North Carolina State University Animal Research Committee.


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  		TABLE 1 Basal diet1

 
    Biochemical analysis. Elastin was quantitated by measuring desmosine, a cross-link amino acid unique to elastin, in animal tissue. Briefly, the aortas and lungs were removed and fixed in Excell (American Master Tech). For the aorta, a 2-mm biopsy punch was taken from the aortic arch, placed in a 1.5-mL microfuge, and hydrolyzed for 24 h in 0.5 mL of 6 mol/L HCl at 100°C. The hydrolysates were evaporated and redissolved in 500 µL water and assayed by RIA for desmosine (13) and protein (14). Hydroxyproline was measured by amino acid analysis. Aorta wall thickness was measured using a Lasico filar eyepiece (Lasico Instruments).

    Histology. Tissues for histology were fixed in Excell (American MasterTech). Lungs were fixed inflated to 20-cm water pressure. A ring of fixed aorta was removed just below the carotids and dehydrated and embedded in paraffin. Sections (5 µm) of the fixed tissues were stained for elastin with a modified Harts elastic stain and counterstained with tartrazine (15). Histology was performed on 10 aortas and 5 lungs from each group. Immunohistochemistry was conducted using an alkaline phosphatase kit from Lab Vision. The sections for immunohistology of fibrillin were incubated at room temperature with 0.2 µg of porcine pancreatic elastase for 10 min prior to incubation with the primary antibody. We did not counterstain to allow for a clearer view of the fibrillin arrangement. The monoclonal antibody for fibrillin-1 (FBN1) and FBN3 were obtained from Lynn Sakai, Shriners Hospital, Portland, OR, and the monoclonal antibody for FBN2 was obtained from Brenda Rongish, University of Kansas Medical Center, Kansas City, MO. For electron microscopy, the lungs and descending thoracic aortas from 2 aortas and lungs were cut into small pieces and fixed in 2% glutaraldehyde in 0.1 mol/L cacodylate buffer containing 2.5% tannic acid, postfixed with 1% osmium tetroxide in the same buffer, dehydrated in ethanol, and embedded in Epon. This preparation gave a high contrast of elastin when thin sections were stained with uranyl acetate and lead citrate.

    Mechanical studies. The aortas were cut into rings 1.5–1.8 mm wide, mounted through wire loops, and clamped into test grips. Aortic rings were mechanically tested in PBS (pH 7.3) at 22°C using a Biosyntech Mach-1 test apparatus (see online supplemental files for detailed methodology of mechanical testing).

    RT-PCR. Total RNA was extracted from aortas using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions and resuspended in diethyl-pyrocarbonate-treated water. The concentration and quality of the RNA were determined by measuring the absorbance at 260 nm and by agarose gel electrophoresis, respectively. Gene expression measurements for a number of chicken genes were determined using real-time quantitative PCR. Primers were designed using Beacon Designer Tm software for SYBR green detection (see online supplemental files for detailed conditions of RT-PCR).

    Statistics. All values are means ± SD except the RT-PCR values, which are expressed as the means ± SE. Statistical analysis of the mechanical test results was done with Statview (Abacus Concepts) using ANOVA and Fisher's protected least significant difference post hoc test. Statistical analysis of the gene analysis and matrix data was performed using a computer software package (InStat, GraphPad). Significance was determined by Student's t test.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Iron deficiency. The body weight of the chicks fed the low-iron diet was less than (P < 0.01) either the moderate-iron group or the controls (Table 2). Hematocrits were lower in both the low- and moderate-iron groups (P < 0.01) compared with the control group. The anemic chicks were lethargic and there was 30% mortality during the 3-wk experimental period. The aorta of the chick is unusually large and thick for the size of the animal and upon removal, control aortas were quite rubbery or elastic. In contrast, the aortas from the iron-deficient chicks were thinner, flaccid, and much less elastic. However, unlike our previous findings with copper-deficient chicks, the vessels were quite strong and showed no evidence of aneurysms.


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  		TABLE 2 Body weights and hematocrits of 3-wk-old chicks fed varying levels of dietary iron1

 
    Biochemical analysis. The desmosine concentrations of aorta and lung did not differ among the groups (Table 3), indicating that they had similar elastin concentrations. Hydroxyproline analyses of the aorta showed that the low-iron group tended to have more collagen (91.8 ± 8 nmol/mg protein) than the control group (81.3 ± 18 nmol/mg protein) (P < 0.12); however, the differences were not significant.


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  		TABLE 3 Desmosine levels in the aorta and lung of 3-wk-old chicks fed varying levels of iron1

 
    Light microscopy. The aorta from control chicks (Fig. 1A) illustrates the uniform and orderly arrangement of elastic fibers throughout the wall of the vessel. In contrast, the iron-deficient aorta wall (Fig. 1B) was much thinner and the elastic fibers appeared wavy and disordered throughout the vessel. The appearance of elastic fibers within the walls of normal chick lung air capillaries is illustrated in Figure 1C. Color was inverted in Photoshop to better illustrate the individual elastic fibers in white. In many areas of the iron-deficient chick lung (Fig. 1D), there appeared to be fewer and thinner elastic fibers, whereas other areas appeared normal. In some locations, the elastic fibers were diffuse and disordered and the air capillary walls were generally thicker than controls. Immunohistology for FBN3 in control chick aorta indicated the presence of large amounts of FBN3, mostly arranged as fibrillar structures parallel to the elastic fibers in the media and adventitia (Fig. 1E). In contrast, FBN3 in the aorta of iron-deficient chicks was present in much lower amounts and was patchy and fragmented in appearance (Fig. 1F). The immunoreaction for FBN2 was very intense and fibrillar in appearance throughout the vessel wall, but amount or appearance did not differ between the control and iron-deficient groups (data not shown). Immunoreaction for FBN1 was more diffuse than either FBN2 or FBN3 and we not able to distinguish differences between the control and iron-deficient aortas (data not shown).


Figure 1
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  		FIGURE 1  Light microscopy of aorta (A,B,E,F) and lung (C,D) from 3-wk-old chicks fed low-iron (B,D,F) and control (A,C,E) diets. Control aorta stained with Harts elastic stain (A) compared with an iron-deficient aorta (B). Notice the wavy appearance of the elastin lamellae in the iron-deficient aorta. A section of a control lung (air capillaries) stained with Harts elastic stain (C) and an iron-deficient lung (D). Elastic fibers appear as white fibers against the darker background due to color inversion. Immunostaining for FBN3 in control aorta (E) and iron-deficient aorta (F). Immunopositive material is red. Magnification, 480x.

 
    Electron microscopy. Electron microscopy revealed that both lungs and descending thoracic aortas of 3-wk-old chickens fed an iron-deficient diet displayed a peculiar spatial organization of cells and extracellular matrix that differed from their counterparts fed the control iron-rich diet. The striking and common feature detected in both lungs (Fig. 2A,B) and aortas (Fig. 2C–F) of low-iron chicks was the presence of vacuolated smooth muscle cells that were surrounded by loose extracellular matrix. This loosely organized matrix consisted of dispersed microfibrillar network and was characterized by the lower-than-normal content of collagen and elastic fibers. In lungs, elastic fibers were short and appeared not fully developed. They consisted of parallel microfibrils decorated with only small patches of electron-dense elastin (Fig. 2B). In contrast, elastic fibers in lungs of the control chicks were much longer and contained a continuous electron-dense elastin core, characteristic of the mature fibers (Fig. 2A). In aortas, the influence of iron deprivation on the organization of the extracellular matrix was even more pronounced. Whereas aortas of control chicks demonstrated well-organized concentric fenestrated elastic lamellae alternating with layers of elongated and tightly attached smooth muscle cells (Fig. 2C,E), aortas of the low-iron chicks (Fig. 2D,F) contained widely dispersed, vacuolated smooth muscle cells that seldom attached to each other and did not form continuous layers. These cells produced sparse fragments of elastin lamellae that appeared to be well polymerized and properly placed on the microfibrillar network, but those fragments (likely produced by different cells) were too short to join each other and form the normal concentric membranes.


Figure 2
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  		FIGURE 2  Electron microscopy of lungs (A,B) and descending thoracic aortas (C–F) from 3-wk-old chicks fed control (A,C,E) or low-iron (B,D,F) diets.

 
    Mechanical studies. Two modulus values were calculated for the whole aortic tissue: 1) a modulus in the elastic region of the curve between strains of 0.2 and 0.4 mm/mm; and 2) a maximum modulus, which was the steepest slope on the curve. The modulus values of the elastic region generally increased from the low-iron group to the control group (Table 4). The maximum modulus tended to be higher in the aortas from the chicks fed the low-iron diet compared with the moderate (P < 0.08) and control (P < 0.12) groups. The groups did not differ in yield stress, yield strain, and energy loss, measures that are independent of cross-sectional area.


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  		TABLE 4 Elastic modulus and strain for whole aorta of 3-wk-old chicks fed varying levels of iron1

 
In contrast to the intact aortas, rings of the cyanogen bromide (CNBr)-digested, purified elastin had a higher yield strain in the aortas from the chicks fed the low-iron diet than those from the chicks fed the moderate-iron diet (Table 5) (P < 0.05). The low-iron and control groups did not differ. There was a lower energy loss in the aortas from the low-iron group than either the moderate-iron or control groups (P < 0.05). The modulus of the aortas of the low-iron group was lower (less stiff) than the moderate-iron group (P < 0.05).


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  		TABLE 5 Elastic modulus and strain for CNBr-digested aorta of 3-wk-old chicks fed varying levels of iron1

 
In terms of total collagen and elastin, there was significantly less tissue supporting the load in the aortic rings of the low-iron group than in either the moderate-iron or control groups (P < 0.05) (Table 6).


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  		TABLE 6 Amount of collagen and elastin per unit length of aorta from chicks fed varying levels of iron1

 
    Gene analysis. Gene expression for the elastin gene was greater in chicks given the moderate level of iron supplementation compared with that in both the low-iron (P < 0.05) and control groups (P < 0.01) (Table 7). This parallels the finding of Bunda et al. (12), who showed that an intermediate level of iron stimulated elastin production in cultured skin fibroblasts to a greater extent than did lower or higher levels of iron. Only 2 genes were dramatically downregulated in response to low levels of iron; these were the FBN1 and FBN3 genes, which exhibited more than a 95% reduction in activity. Other genes unresponsive to dietary levels of iron included myosin light and heavy chains, the insulin like growth factor 2, epidermal growth factor, epidermal growth factor receptor, and activating transcription factor 3.


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  		TABLE 7 Effect of dietary iron levels on gene expression of 3-wk-old chicks fed varying levels of iron12

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Previous in vitro studies suggested that low and high levels of iron could adversely influence the formation of elastic fibers (12). We investigated the effects of a low-iron diet on elastic fiber formation in the aortas and lungs of young chickens. After 3 wk of treatment, the chicks experienced the expected weight loss and anemia associated with iron deficiency but showed no gross pathology such as aortic aneurysms that could be due to the effect of iron deficiency on elastic fibers.

Biochemical analysis for desmosine indicated that the elastin content in the aortas of chicks fed the low- or moderate-iron diets did not decrease compared with the control diet when based on a unit protein basis. Desmosine cross-links are only present in elastin in animal proteins and are a precise reflection of the quantity of mature elastin in a tissue. This assumes, however, that the cross-linking process itself was not altered. Desmosine analysis of the elastin purified by CNBr extraction for the stress-strain studies confirmed that the desmosine cross-linking did not differ in the iron-deficient and control elastin.

Because iron is required for prolyl hydroxylase activity and the conversion of proline to hydroxyproline, one might anticipate decreased collagen content of the iron-deficient aortas. This was not the case in our study, however, as the hydroxyproline content of the aortas did not differ among groups.

Histological examination of the aortas and lungs showed only subtle differences in the elastic fiber network of the iron-deficient chicks but showed no evidence of a loss of elastin or fractured elastic fibers. The most notable observation was the very wavy nature of the elastin lamellae throughout the vessel wall in the low-iron group, which may have had some influence on the stress-strain performance. The apparent diminished elastin content of the air capillaries of the lung could be due to the thicker air capillary walls and more diffuse elastic fiber distribution.

Immunolocalization demonstrated all 3 fibrillins (FBN1, FBN2, and FBN3) in the aortas of the control chicks were abundantly expressed. The reason why FBN3 was the only fibrillin severely affected by iron deficiency is not known. FBN3 has been shown to be highly expressed in fetal and developing tissues in humans (16). A similar study has not been conducted to show the importance or age-related changes in the fibrillins of the developing chicks. Electron microscopy confirmed that, in both aortas and lungs of iron-deprived chickens, there was a strikingly loose extracellular matrix consisting of dispersed and unorganized microfibrils and elastin. In aortas, the elastic lamellae were fragmented but properly oriented; however, those fragments were apparently too short to join each other and form the normal concentric membranes. In lungs, these structures looked even less developed and consisted of small patches of electron-dense elastin, decorating scarce and shorter than normal microfibrils. Together, the morphologic observations strongly support the data obtained from gene expression analysis, indicating that the iron deficiency contributes to a disproportional deposition of the major components of elastic fibers and, as a consequence, alters the normal assembly of these most complex components of the extracellular matrix.

A number of factors contribute to the mechanical properties of arteries, including the amount of elastin and/or collagen in the vessels, the cross-link density, and the ultrastructure of the matrix. The differences in mechanical properties of the vessels in our experimental groups cannot be explained by changes in the relative amount of elastin or collagen in the vessels, because these did not differ among the groups. However, only one-half the amount of total matrix material bore the load in the aortas of chicks fed the low-iron diet. The cross-link density in elastin did not differ among groups and was, therefore, not responsible for the differences in mechanics. The decreased modulus in the low-iron group, as compared with the moderate-iron group, was significant and suggests a change in the elastin, which gives a less stiff matrix and increased extension before break. This suggests that the quality or the organization/architecture of the elastin, collagen, and microfibrils bearing the load was altered in the iron-deficient aortas compared with the iron-supplemented, control chickens.

Despite the structural changes of elastic fibers detected in lungs and aortas of neonatal chicks fed the low-iron diet, low levels of dietary iron did not downregulate elastin gene expression compared with the control group. Intermediate levels of iron actually upregulated the elastin gene compared with control chicks, an observation also noted in the cell culture studies (12). These results, together with the fact that the net amount of insoluble elastin did not differ in the inspected tissues, indicate that fluctuations in elastin message levels in young chicks may not be a major factor responsible for the structural changes of elastic tissue elements detected in our experimental model. Instead, we found that low levels of dietary iron markedly downregulated expression of genes encoding FBN1 and FBN3. The fibrillins are crucial components of the microfibrillar scaffold that is prerequisite to the normal elastic fiber assembly (17–22). The fact that both low levels of dietary iron downregulated the fibrillin genes suggests that relative high dietary levels of iron are required to maintain adequate fibrillin levels. These results allow us to speculate that the relative iron deficiency created in our experimental model interfered with the formation of new microfibrils, thereby affecting the postnatal growth and remodeling of elastic lamellae and fibers and contributing to structural and mechanical complications of lungs and vessels.

The significantly increased ferritin gene expression in the aortas of chicks fed the low-iron diet was unexpected, because ferritin and iron content are usually positively correlated (23). However, in this study, the chicks were anemic and the organs may have been in a state of hypoxia, which is known to have deleterious effects on vascular tissue (24–27). Evidence that this was true was the increase in the expression of the hypoxia-induced factor gene in the aortas of the chicks fed the low-iron diet.

In summary, our data suggest that inadequate iron intake during the neonatal period results in a structural and functional compromise of elastic fibers. Our novel observation that lowering the dietary iron intake was associated with downregulation of FBN1 and FBN3 gene expression and deposition of FBN3 in tissues opens a new direction for future studies aimed at the molecular mechanism by which iron could modulate microfibril production.


   FOOTNOTES
 
1 Supported by Flight Attendants Medical Research (B.S.) and Heart and Stroke Foundation of Ontario (A.H.). Back

2 Author disclosures: C. H. Hill, C. M. Ashwell, S. J. Nolin, F. Keeley, C. Billingham, A. Hinek, and B. Starcher, no conflicts of interest. Back

3 Supplemental Table 1 and other supplemental data are available with the online posting of this paper at jn.nutrition.org. Back

Manuscript received 12 March 2007. Initial review completed 5 April 2007. Revision accepted 14 June 2007.


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TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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