Effect of hypertension on fibronectin expression in the rat aorta.

Interactions between extracellular fibronectin and vascular cells are thought to influence the phenotype of those cells. To determine if changes in fibronectin expression accompany the phenotypic changes of vascular tissue characteristic of experimental hypertension, steady state mRNA levels for fibronectin were determined in aortae of normotensive and hypertensive rats. A 3-6-fold increase in fibronectin mRNA was observed in aortic tissue of hypertensive rats following 3 weeks of treatment with deoxycorticosterone and salt, whereas if rats were treated only with deoxycorticosterone or salt alone, no changes occurred. The changes were reversed by normalization of blood pressure. The increases observed were localized to aorta and not to the periaortic tissue. Angiotensin II infusion using osmotic minipumps also caused an increase in fibronectin expression. Age-dependent increases in aortic fibronectin mRNA occurred in several rat strains, and the combined effects of hypertension and aging were greater than either variable alone. A clear distinction between the expression of fibronectin mRNA and that for collagen or tropoelastin were found in hypertensive and aging models. Aortic fibronectin was also increased in the hypertensive rats as determined by Western blot analysis. The findings indicate that elevation in blood pressure increases fibronectin expression in rat aorta and suggest that such changes may influence the aortic cellular responses to hypertension.

Fibronectin is a widely distributed glycoprotein localized in the extracellular matrix of various cell types and has been implicated functionally in the regulation of several cellular processes, including adhesion, differentiation, motility and transformation (reviewed in Refs. 1 and 2). In vascular tissue, fibronectin has been demonstrated immunohistochemically in aorta and other large vessels (3)(4)(5), but the insoluble nature of the extracellular matrix has made quantitation difficult (6)(7)(8). Interactions between fibronectin and cultured vascular smooth muscle cells induced a phenotypic change from a contractile to a synthetic state, and it has been suggested that the cellular changes that occur during atherosclerosis may be due to fibronectin-cell interactions (9). A recent study has shown that alternatively spliced variants of fibronectin are present in aortic tissue, and that following vascular injury, proliferating smooth muscle cells switch from one variant to another (10). This phenomenon was observed using both tissue culture and in uiuo animal models (10). Transforming growth factor-p (TGF-/3)' has been shown to influence fibronectin expression in fibroblasts and endothelial cells, and it was suggested that several effects of TGF-P on cellular growth were mediated by changes in fibronectin production (11)(12)(13)(14)(15). We reported recently on the increased steady state mRNA level of TGF$ in rat aorta associated with experimental hypertension induced by treatment with deoxycorticosterone and excess salt (16). The present study was designed to determine if changes in fibronectin expression occurred following experimental hypertension in the rat. tion was conducted for 18 h at 65 "C using cDNA probes labeled by the random hexamer priming procedure (19) in a hybridization buffer of the following composition: 745 mM NaCI, 50 mM NaH,PO+ 5 mM EDTA, 0.05% pyrophosphate, 3% sodium dodecyl sulfate, 10% dextran sulfate, and 200 pg/ml sonicated herring sperm DNA. After hybrization, the nylon membranes were washed 4 times for 5-10 min at 55 "C with 0.5% sodium dodecyl sulfate and 1 X, 0.5 X, 0.25 X, and 0.1 X standard saline citrate (SSC) (1 X SSC is 0.15 M NaCl. 0.015 M sodium citrate, pH 7.0), respectively. The nylon membranes were then exposed to preflashed x-ray films (Kodak X-Omat AR) between two intensifying screens for 3 h to 5 days at -70 "C. Laser densitometry (Molecular Dynamics model 300A) was used to quantitate the relative signal intensity of the bands obtained. Equal amounts of RNA in each lane were confirmed using a cDNA probe for P-actin and by visual examination of ribosomal RNA using ethidium bromide staining.

Western
Blot Analysis of Aortic Fibronectin-Aortie from control or hypertensive animals were carefully cleaned and cut into rings following the general procedures described previously by us (24). Aortic rings from a single rat aorta were homogenized using a motordriven glass-glass apparatus in phosphate-buffered saline containing aprotinin (1.2 mg/ml), leupeptin (10 mM), pepstatin A (1 mM), and phenylmethylsulfonyl fluoride (1 mM), at a 20~1 ratio of buffer volume to tissue wet weight. The homogenate was centrifuged at 25,000 X g for 20 min. The pellet was resuspended in 4% sodium dodecyl sulfate (SDS), the resuspension heated at 100 "C for 4 min, and then centrifuged at 12,000 x g for 5 min. The SDS extract was removed and saved. All samples were stored at -70 "C. Protein concentrations were obtained by using the bicinchoninic acid protein assay reagent kit by Pierce Chemical Co.
Proteins were analyzed by SDS-polyacrylamide gel electrophoresis as described by Laemmli (25) using Mini-Protean II Dual Slab Cell apparatus (Bio-Rad). For Western blot analysis Mini Trans-Blot Cell apparatus (Bio-Rad) was used. Transfer onto nitrocellulose was done at 4 "C for 18 h in 20 mM Tris, 200 mM glycine, and 20% methanol. Immunological detection was performed using a goat anti-human polyclonal antibody to fibronectin (Sigma) following pretreatment of the nitrocellulose with 10% Carnation evaporated milk. Subsequent analysis utilized an anti-goat IgG horseradish peroxidase conjugate (Sigma) as a second antibody, and chemiluminescence emitted from luminol oxidized by peroxidase as a detection method (ECL Western blotting detection system, Amersham, United Kingdom). The first antibody was used at a dilutuion of 1:3,000 and the second antibody at a 1:75,000 dilution. The general protocol was similar to that described in the instructions provided by the supplier.
Statistical Analysis-For the statistical analysis of blood pressure differences between groups, the unpaired Student's t test or one-way analysis of variance followed by a multiple comparison test was performed.

RESULTS
The DOG/salt model of experimental hypertension in the rat involves uninephrectomy followed by administration of DOC, a steroid with mineralocorticoid activity, and an excess of dietary salt, given by substituting saline for water. Fig. IA shows a Northern blot analysis for fibronectin using aortic RNA taken from rats treated with or without DOC and salt. Each lane contains 20 pg of total RNA from pooled aortic tissue of 3-4 rats that were treated in an identical manner at the same time. Mean and standard deviation for systolic blood pressure, and the number of rats pooled for each RNA sample are shown below each lane. There was no change in steady state fibronectin mRNA levels when uninephrectomized rats were untreated for 3 weeks (UN); treated with salt but no DOC for 3 weeks (UPJ/salt); treated with DOC but no salt for 3 weeks (UN/DOC); or when rats with both kidneys intact were given DOC but no salt for 3 weeks (DOC). However, when uninephrectomized rats were given both DOC and salt treatment for 3 weeks, a 6.1-fold increase in fibronectin Mean SSP 136 154 129 12,191* 16&14(4) ' (3) (3) (4) (4)  Each lane contains 20 rg of total aortic RNA obtained from animals treated as designated for 3 weeks. A, the effect of different treatment regimens on the steady state mRNA level of fibronectin and fl-actin in aorta. UN, uninephrectomy alone; UN/salt, uninephrectomy and administration of 0.9% NaCl (saline) in drinking water; lJN/DOC, uninephrectomy, implantation of a DOC pellet, andlow sodium diet; DOC, implantation of a DOC pellet alone; UN/DOG/salt, uninephrectomy, implantation of a DOC pellet, and saline-drinking; Regression, 6-week administration of a diuretic (chlorothiazide) and a low salt diet after a 3-week UN/DOG/salt treatment. Mean and SD. of the svstolic blood pressure (SBP), the number of rats pooled for each RNA sample, and the statistical significance of the blood pressure increase after the treatment are shown at the bottom of each lane. *p < 0.05. Mean systolic blood pressure for the last lane (Regression) was I88 mm Hg at the end of the UN/DOC/ salt treatment and 118 mm Hg at the end of the diuretic/low salt treatment. B, tissue-specific increase of steady state mRNA level of fibronectin in aorta. The strongest increase of fibronectin mRNA was present in aorta after a 3-week UN/DOG/salt treatment. Periaortic tissue loosely adhering to aorta showed a minor increase of fibronectin mRNA, and no change in interscapular brown fat. kb, kilobases. mRNA was seen (U~/DOC/salt). This increase was associated with higher systolic blood pressure (191 mm Hg) than in the other groups, although animals given salt but no DOC (LUV/salt) had mild increases in systolic blood pressure (154 mm Hg) but no obvious change in fibronectin expression. The last lane contains RNA from uninephrectomized rats treated with DOC and salt for 3 weeks followed by a period of reversal of hypertension by cessation of DOG/salt treatment and institution of a low salt diet and diuretic therapy (500 mg/ liter chlorothiazide included in the drinking water) for 6 additional weeks. The absence of a strong signal for fibronectin mRNA indicates that reversal of DOG/salt treatment was associated with a reversal in the increased levels of fibronectin mRNA. Fig. 1B contrasts the intensity of the signal for fibronectin mRNA from aortic tissue with that of periaortic tissue and brown adipose tissue, using samples from control and hypertensive animals. Equivalent amounts of total RNA (20 pg) were applied to each lane prior to Northern blot analysis. The strongest signals were obtained using aortic tissue, and the 6fold increase in aortic fibronectin mRNA seen following DOC/ salt hypertension was clearly greater than the IA-fold increase seen in periaortic tissue. Thus the increased steady state mRNA levels can not be accounted for by the relatively small amount of contaminating periaortic tissue that is difficult to completely remove from aortae prior to isolation of RNA.
Steady state mRNA levels for fibronectin, tropoelastin, collagen, and fi-actin in aortae of animals made hypertensive by two different models of experimental hypertension, angiotensin II infusion and DOG/salt treatment, are shown in Fig.  2 injection of angiotensin II (133 ng/min) using an osmotic minipump for 3 and 10 days, respectively. Fibronectin mRNA levels were elevated in both cases when compared to a representative control sample taken from rats infused only with vehicle (lane 1). When the RNA was rehybridized using cDNA probes for either tropoelastin or collagen type I, major components of the extracellular matrix, there were no consistent changes in steady state mRNA levels for those substances. However, fibronectin mRNA was consistently increased in all RNA samples tested, showing a 2.0 + 0.3-fold increase (mean + S.E., n = 6) when comparing six separate RNA samples from 17 rats treated with angiotensin II uersw four separate RNA samples obtained from 9 control rats. Thus, the increase in fibronectin mRNA seen following angiotensin II infusion was relatively specific and not associated with a general increase in gene expression of all components of the extracellular matrix. Fig. 2 (lanes 5 and 6) contains aortic RNA from animals subjected to DOG/salt treatment for 2 and 3 weeks, respectively. Fig. 2 (lane 4) contains RNA from uninephrectomized control rats that were untreated for 2 weeks following uninephrectomy. When hypertension was produced by DOG/salt treatment, there was an increase in steady state mRNA levels for aortic fibronectin. Densitometric analysis comparing five separate RNA samples from 15 DOG/salt-treated rats uersus four separate RNA samples from 14 uninephrectomized control rats showed a 5.1 + 1.5-fold increase (mean + S.E., n = 5) following 2 and 3 weeks of treatment. When the same RNA samples were rehybridized with cDNA probes for tropoelastin or collagen type I, increased steady state mRNA levels were Aortic Fibronectin 21937 not seen; rather, there was a decrease in tropoelastin mRNA levels and no obvious change in collagen type I mRNA. In one study using aortic RNA from rats treated with DOG/salt, we did observe a moderate increase in collagen mRNA, but that finding was not consistent whereas we invariably observed increased fibronectin expression. The increase in fibronectin mRNA levels was also characteristic of a genetic model of hypertension. Fig. 3A shows measurements of fibronectin mRNA in aortic samples from SHR and age-matched WKY. Densitometric comparisons indicated an aproximate 2-fold increase in the SHR at all ages past 5 weeks, whereas the youngest group showed a 3.3-fold greater signal in the WKY than the SHR. In both strains, increasing age was associated with an increased signal. Again, when the same RNA samples were hybridized with cDNA probes for collagen or tropoelastin, a clear dissociation between the regulation of steady state mRNA levels between fibronectin and either collagen or tropoelastin was apparent. The steady state mRNA level of /3-actin was slightly and - consistently less in younger rats, although the ethidium bromide staining of the gel showed equivalent loading of RNA among samples. In Fig. 3B, the effect of age on fibronectin expression was extended to the Wistar rats and to Fischer rats, a strain often used for aging studies (26). In both strains, increased fibronectin mRNA was seen with increased age. In the middle panel of Fig. 3B, a comparison between aortic RNA from age-matched Wistar, WKY, and SHR showed that hypertension may exacerbate further the changes normally associated with age. Fig. 4

DISCUSSION
An increase in steady state mRNA and protein for fibronectin in the rat aorta was found following induction of two forms of experimental hypertension.
In addition, aortic fibronectin mRNA was shown to increase with age in several rat strains and these age-related changes appeared to be enhanced in the presence of hypertension.
It has been suggested that the changes that occur normally in the vessel wall with age are accelerated by experimental hypertension (27,28). Increased fibronectin expression appeared to be associated with aging in four rat strains studied, and hypertension appeared to accelerate age-related changes further, based on the comparative data between 40-week-old Wistar, WKY, and SHR rats shown in Fig. 3B. The contrast between fibronectin and other major components of the aortic extracellular matrix was most evident when compared in the SHR where the marked increases in fibronectin mRNA were clearly distinguished from the decreased levels of mRNA for either collagen or tropoelastin.
In a recent study, changes in fibronectin expression were documented in cultured vascular smooth muscle cells that were converted from a contractile to synthetic phenotype (10). In addition, the appearance of an alternatively spliced variant of fibronectin was found in vivo in the aortic intima following the induction of intimal lesions by balloon injury in the rat or atherosclerotic lesions in man (10). The in vivo hypertensive models that were used in the present study are known to produce medial changes such as hypertrophy and polyploidy (27,29,30). Our observations suggest that the increase in aortic fibronectin mRNA was localized to aorta, and not to the periaortic tissue, which includes adipocytes and several other cell types (31). Other studies on gene expression in vascular tissue showed that pulmonary hypertension induced increased elastin expression in adventitial fibroblasts, perhaps stimulated by a paracrine mechanism originating from vascular smooth muscle (32). Studies on expression of the various components of the renin-angiotensin system in vascular tissue have implicated the periaortic adipocyte as the major producer of angiotensinogen (31). The mechanism responsible for increased aortic fibronectin expression in hypertensive animals is difficult to assess. TGF-/3 has ben shown to regulate the expression of fibronectin and other components of the extracellular matrix in fibroblasts and aortic endothelial cells (11)(12)(13)(14)(15). However, studies with cultured vascular smooth muscle cells have shown that TGFp does not induce fibronectin (33). In addition to TGF-P, substances shown to influence fibronectin expression in cultured cells include glucocorticoids (34), CAMP (34), interleukin-6 (35), epidermal growth factor (36), platelet-derived growth factor (36,37), glucose (38), heparin (33, 39), and tumor necrosis factor (40). Responses to these substances differ markedly depending on the cell type and culture conditions. There is little evidence for any of these agents influencing fibronectin expresssion in cultured vascular smooth muscle cells and no evidence to date for any hormone or growth factor influencing aortic fibronectin biosynthesis in vivo.
The functional significance of altered aortic fibronectin expression in the hypertensive rat also is difficult to assess at present. Hypertrophy is a common response of vascular smooth muscle to experimental hypertension or to vasoactive agents such as angiotensin II or norepinephrine. An early event in the induction of hypertrophy is thought to be a shift in the cell cycle from Go to G, and fibronectin has been implicated as one of the early response genes that may be rapidly expresssed when cells are exposed to certain growth factors that initiate cell cycle changes (36). However, despite the multiplicity of functions that have been attributed to fibronectin in both physiological and pathological processes, it is not possible at present to establish with certainty what role fibronectin has in vascular tissue of either normotensive or hypertensive animals. The consistent and long-lived increase in aortic fibronectin mRNA levels in aging or hypertensive rats suggest a possible role for fibronectin in mediating the morphological and functional changes that accompany those conditions, but additional studies concerning the localization of fibronectin in aorta and relative contribution of specific isoforms of fibronectin will be necessary to determine if such a role exists.