Articles

Angiotensin II Induction of Osteopontin Expression and DNA Replication in Rat Arteries

Denis deBlois, Donna M. Lombardi, Enming J. Su, Alexander W. Clowes, Stephen M. Schwartz, Cecilia M. Giachelli
https://doi.org/10.1161/01.HYP.28.6.1055
Hypertension. 1996;28:1055-1063
Originally published December 1, 1996

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Abstract

We recently identified the adhesive protein osteopontin as a novel smooth muscle cell product overexpressed in rat developing neointima and human atheroma. Although osteopontin is a candidate stimulant for intimal lesion progression because of its chemotactic and calcium binding functions, factors controlling osteopontin expression in arteries remain poorly defined. In vitro, smooth muscle cell expression of osteopontin is associated with cell cycle transit or alterations in cell phenotype, and it is increased by angiotensin II (Ang II) stimulation. In the present studies, we investigated both osteopontin expression and DNA replication in the arterial wall in response to chronic Ang II infusion in vivo. Rat carotid arteries with or without intimal thickening (induced by balloon catheterization) were examined. Ang II (250 ng/kg per minute) or vehicle was coinfused with bromodeoxyuridine (to label replicating DNA in vivo) for 2 weeks beginning 4 weeks after injury. With Ang II, smooth muscle cells overexpressed osteopontin as shown by protein immunohistochemistry, in situ hybridization, and Northern blot analyses. Osteopontin mRNA levels were increased markedly (approximately fivefold) in the normal artery media and injured artery neointima, but levels remained low in the injured artery media, in positive correlation (R2=0.88, P<.001) with DNA replication in the smooth muscle layers, further suggesting that osteopontin may be a growth-associated, phenotype-dependent gene for smooth muscle cells. However, osteopontin expression in neointima was not restricted to areas showing DNA replication, suggesting a nonobligatory association. Ang II induced severe hypertension. Arterial osteopontin expression was increased also by chronic catecholamine infusion, a model of vascular growth stimulation showing labile pressure elevations. Osteopontin induction in smooth muscle cells may contribute to Ang II–dependent intimal lesion progression and vascular remodeling events associated with renovascular diseases or hyperadrenergic disorders.

Originally described as a bone extracellular matrix protein, osteopontin is a low-affinity, high-capacity calcium binding phosphoprotein that contains the integrin receptor binding amino acid sequence arginine-glycine-aspartate.1 We recently identified osteopontin as a developmentally regulated novel smooth muscle cell product2 that is expressed in strong association with intimal lesions in the arterial wall,3 where it is colocalized with dystrophic calcification, inflammation, and myofibrous regions,4 5 6 and that interacts with αvβ3-integrin to stimulate adhesion or chemotaxis in smooth muscle and endothelial cells.7 8 In human atherosclerotic plaque, smooth muscle–derived foam cells and macrophages are major sources of osteopontin expression.4 5 9 Osteopontin expression by smooth muscle cells is low in normal rat arteries but transiently elevated during the migratory and proliferative phases of neointima formation, ie, during the first 3 weeks, after balloon injury.2 3 Recent studies with smooth muscle cells cultured from normal arterial wall have suggested that overexpression of osteopontin is associated with cell cycle transit in response to serum stimulation10 or with the loss of a differentiated contractile phenotype with time in culture.11 However, factors regulating osteopontin expression in the arterial wall in vivo remain poorly defined.

Angiotensin II (Ang II), a vasopressor peptide implicated in the rat arterial injury response, is one of the most potent known inducers of osteopontin expression in cultured smooth muscle cells.3 Thus, we speculated that osteopontin may be elevated in the arterial wall during Ang II–induced hypertension. Moreover, Ang II–induced hypertension is associated with increased smooth muscle DNA replication.12 We recently observed that responsiveness to Ang II as a smooth muscle cell mitogen is lost in the injured media as early as 9 weeks after balloon injury, whereas Ang II remains a significant mitogen only in the neointima as late as 6 months after injury.13 Also, cells cultured from the injured artery media at 9 weeks after injury show a reduced proliferative response to serum in vitro compared with cells from the neointima or normal arterial wall.14 Together, these data suggest that smooth muscle cell phenotype may be differently altered in the media and neointima after injury. We designed the present studies to evaluate and localize osteopontin expression in the media and neointima of rat arteries in response to chronic Ang II infusion. We also investigated the possible correlation between levels of DNA replication and osteopontin gene expression in the media and neointima. Ang II was infused continuously into rats during the 5th and 6th weeks after balloon injury to the left common carotid artery. This period was chosen because previous studies have shown that injury-induced smooth muscle cell replication and osteopontin expression are reduced to uninjured levels by 4 weeks after injury.3 In addition to Ang II–infused rats, in which hypertension was severe, we also examined osteopontin expression in an angiotensin-independent rat model of labile hypertension15 induced by catecholamine infusion. The present results provide further evidence that osteopontin may be a growth-associated, phenotype-dependent gene for smooth muscle cells.

Methods

Surgical Procedures

Formation of a neointima was induced in the left common carotid artery of 24 male Sprague-Dawley rats (400 to 450 g, Zivic-Miller Laboratories, Inc, Allison Park, Pa) by arterial dilatation with a balloon embolectomy catheter (size 2F, Fogarty) as described previously12 and in accordance with institutional guidelines. Rats were anesthetized with a single intramuscular injection of ketamine (80 mg/kg) and xylazine (4 mg/kg). At the beginning of the 5th week after injury, rats were infused continuously for 2 weeks with either Ang II (250 ng/kg per minute) or Ringer's lactate vehicle via osmotic pumps implanted subcutaneously (n=12 rats per group). In experiments designed to measure the Ang II effect on smooth muscle growth, the rats (n=6 per group) also received via a separate pump a continuous coinfusion of 5-bromo-2′-deoxyuridine (BrdU, 0.8 mg/kg per day) to label cell nuclei undergoing DNA replication at any time during the 2-week infusion period. Systolic pressure was monitored by tail-cuff plethysmography before and during drug infusion. Rats were weighed at the time of vascular injury, at the time of pump implantation, and again at the time of death.

At the end of the drug infusion period, all rats received a bolus injection of Evans blue (80 mg/kg) to stain the segment of carotid artery that lacked endothelial cell regrowth (typically the middle two thirds of the carotid artery length). Rats were killed by pentobarbital overdose 15 minutes after Evans blue. Arterial segments for histological studies were obtained from the middle portion of the uninjured carotid artery as well as from the blue (without endothelium) segments of the injured carotid artery, were fixed by immersion in phosphate-buffered 4% paraformaldehyde or in MethaCarnoy's fixative, were processed in graded alcohol and xylene according to routine histological procedures, and were embedded in paraffin. Arterial specimens were also obtained from the thoracic aorta (uninjured) at the level between the third and fourth intercostal arteries. Tissue sections (5 μm) were obtained for immunohistochemical or in situ hybridization studies as described below.

Northern Blot

A subset of 12 rats infused with either Ang II or Ringer's lactate (n=6 per group) was used as a source of vascular tissue for osteopontin gene expression studies by Northern blot analysis of total smooth muscle RNA. Briefly, arteries were isolated, cleaned free of adventitia and endothelium, and immediately snap-frozen in liquid nitrogen until further processing. In the injured carotid artery, only the blue segment was used. Before freezing, the neointimal layer was peeled from the underlying medial tissue with microforceps under a dissecting microscope for separate processing of the two smooth muscle layers. Tissues were ground in liquid nitrogen, and total mRNA was extracted by the guanidinium isothiocyanate method as described previously.16 Northern blot analysis of osteopontin mRNA was performed as described previously with the 2B7 cDNA probe (multiprime-labeled) coding for the rat osteopontin gene.2 3 The level of osteopontin gene expression in the different smooth muscle layers was normalized over the signal for the 28S rRNA (end-labeled probe) by quantifying the radioactivity associated with the mRNA species with the use of a PhosphorImager (Molecular Dynamics).

In a previous study of catecholamine-mediated cardiovascular effects, we infused rats for 2 weeks with norepinephrine (2.5 mg/kg per day), the selective α1-adrenergic agonist phenylephrine (25 mg/kg per day), Ringer's lactate containing 0.02% ascorbate (used as a preservative for catecholamines), or Ringer's lactate alone and observed a significant catecholamine-dependent stimulation of arterial DNA synthesis and increase in systolic pressure lability, resulting in borderline hypertension.15 In the present study, we used the total RNA extracted from the uninjured carotid artery media of these rats (one vessel per rat, six rats per group) to quantify arterial osteopontin expression in an Ang II–independent model of vascular growth and blood pressure regulation.

In Situ Hybridization

In situ hybridizations were performed with 35S-labeled riboprobes on 5-μm paraffin sections of 4% paraformaldehyde–fixed tissue. An approximately 1100-bp portion of the 1.4-kb gene for rat osteopontin was cloned into pBluescript S/K (Stratagene) as described previously.2 DNA was linearized with Bgl I or Mam I, and riboprobes were synthesized with T7 or T3 RNA polymerase (for antisense and sense probes, respectively) in the presence of 35S-labeled [α-thio]UTP (New England Nuclear–DuPont) following a modified method of Wilcox et al.17 Riboprobes were separated from unincorporated counts by passage over G-50 NICK columns (Pharmacia) and were subsequently ethanol-precipitated overnight and resuspended in 300 000 cpm/μL buffer containing 10 mmol/L Tris-HCl and 1 mmol/L EDTA. Slides were prehybridized with hybridization buffer alone at 55°C for approximately 2 hours and then hybridized overnight at 55°C with probe in the hybridization buffer equivalent to 300 000 cpm per section. Washes included treatment with RNase A (20 mg/mL, Sigma Chemical Co) for 30 minutes at 37°C and a stringent wash in 0.1× SSC at 55°C for 2 hours. After being dehydrated and air-dried, slides were dipped in NTB2 emulsion (Eastman Kodak), exposed at 4°C for 14 days, and developed as previously described.17 Hematoxylin was used as counterstain.

Immunohistochemistry

Nuclear incorporation of BrdU was used as a marker for DNA replication during a 2-week drug infusion period. Paraformaldehyde-fixed tissue sections were used for detection of BrdU incorporated into the nuclei of smooth muscle cells as described previously18 with an indirect peroxidase-labeled antibody technique using reagents from a kit (Vectastain Elite, Vector Laboratories). Slides were immersed in hydrogen peroxide (3%) for 35 minutes to quench endogenous peroxidase activity. The chromogen 3,3′-diaminobenzidine (SigmaFast, Sigma) was used for staining of the immunoreactive nuclei. Sections were counterstained with hematoxylin before being permanently mounted under coverslips. The number of smooth muscle cells in the media and intima per cross section was counted under light microscopy. Results are expressed separately for intima and media as the cumulative labeling fraction. A similar double-antibody protocol was used for staining of sections of MethaCarnoy's-fixed carotid arteries for osteopontin with, as primary antiserum, the goat polyclonal antibody OP199 (1:100) raised against rat smooth muscle cell osteopontin8 or staining for the presence of monocyte-macrophages with the monoclonal antibody ED-1 (1:1000, Serotec, Harlan Bioproducts for Science) as a primary antiserum in paraformaldehyde-fixed sections of carotid arteries. We have previously shown by Western blotting that the OP199 antiserum is specific for osteopontin.8 Control sections were incubated with the corresponding nonimmune serum.

Measurement of Vascular Cross-sectional Area

For each vessel, two nonconsecutive cross sections (5 μm) were stained with the elastin fiber–specific stain orcein, and cross-sectional areas were measured by light videomicroscopy with a computerized morphometry system (BioScan). Medial area was defined as the area enclosed between the external and internal elastic laminae, and intimal area was defined as the area between the internal elastic lamina and lumen perimeter.

Statistical Analysis

Values are expressed as mean±SE. Student's t test was used for comparison of corresponding data between the Ang II– and Ringer's-treated groups. In the Ang II experiments, a correlation analysis between smooth muscle DNA replication and osteopontin expression was performed with the mean BrdU-labeling fraction (in normal media, injured media, and neointima; n=6 rats per group) and the corresponding osteopontin mRNA level, as measured by Northern blot analysis of total RNA pooled from a different set of tissues from six rats per group. In the catecholamine experiments, the replication data and osteopontin expression data for correlation analysis were measured in uninjured carotid arteries isolated from the same set of rats. A value of P<.05 was considered statistically significant.

Results

Ang II and Blood Pressure Regulation

Continuous infusion of Ang II (250 ng/kg per minute) caused a significant (P<.05) increase in systolic pressure, from 130±3 mm Hg (n=12) before infusion up to 173±10 on day 6 and 199±5 on day 13. Systolic pressure was not affected in rats infused with Ringer's lactate (124±3 mm Hg before infusion versus 125±3 on day 6 and 134±3 on day 13; n=12).

Ang II–Stimulated Osteopontin Expression in Uninjured Arteries

We previously reported that smooth muscle cells in normal arterial wall express low levels of osteopontin gene and protein.3 We now report that Ang II infusion markedly increased steady-state levels of osteopontin mRNA in normal arterial media by more than fourfold after a 2-week infusion, as shown by Northern blot analysis with the 2B7 cDNA coding for the rat osteopontin gene (Fig 1⇓, top, lanes 1 and 2). Likewise, the thoracic aorta media (uninjured) showed osteopontin mRNA levels that were similar to those in the corresponding carotid artery media (uninjured) from the same rats (data not shown). The increase in osteopontin mRNA in the carotid artery was confirmed by in situ hybridization analysis with an antisense riboprobe generated by transcription of the 2B7 plasmid (Fig 2A and 2B⇓⇓). The increased hybridization signal in carotid artery media from Ang II–infused rats showed a focal pattern of distribution, predominantly around cell nuclei. The sense probe gave no localized signal in any of the arteries examined (not shown). Next, we determined that carotid arteries from Ang II–infused rats showed increased immunoreactivity for the polyclonal goat antiserum OP199 raised against rat smooth muscle cell osteopontin (Fig 3A and 3B⇓⇓). Low-level diffuse staining was present in the media in the Ang II group, but intense patches were also seen near cell nuclei. Staining with the OP199 antiserum was negative in arteries from the control group.

Figure 1.
Figure 1.

Top, Northern blot analysis of osteopontin mRNA levels from normal and injured carotid arteries of rats infused continuously with angiotensin II (250 ng/kg per minute) or Ringer's lactate 5 and 6 weeks after balloon injury to the left carotid artery. Top autoradiogram shows levels of osteopontin mRNA (size, 1.6 kb) in uninjured artery media (lanes 1 and 2), injured artery media (lanes 3 and 4), and injured artery neointima (lanes 5 and 6) after angiotensin II (lanes 2, 4, and 6) or Ringer's lactate (lanes 1, 3, and 5). Between 5 and 15 μg total RNA was loaded per lane, depending on the quantity of material extracted (pool of six rats per group). The osteopontin autoradiogram resulted from a 15-hour exposure of the filter hybridized with the 2B7 cDNA coding for the rat osteopontin gene. Size of the observed bands was estimated on the basis of migration of the 18S and 28S rRNAs (revealed with methylene blue staining). Bottom, autoradiogram shows methylene blue–stained filter with 28S rRNA bands (size, 4 to 4.4 kb). Bottom, Densitometric analysis of osteopontin mRNA levels shown in the autoradiogram normalized to 28S rRNA levels (evaluated by autoradiography). Values are given as ratio of osteopontin to 28S and expressed in arbitrary densitometric units. Boxed number at the base of each column refer to corresponding lane of the autoradiogram.

Figure 2.
Figure 2.

In situ hybridization analysis of osteopontin mRNA levels in normal and injured carotid arteries of rats infused continuously with angiotensin II (250 ng/kg per minute) or Ringer's lactate 5 and 6 weeks after balloon injury to the left carotid artery. The riboprobe was generated from transcription of the 2B7 plasmid. A, Uninjured carotid artery from Ringer's-infused rat; B, uninjured carotid artery from angiotensin II–infused rat, showing increased grains over cells in the media; C, injured carotid artery from Ringer's-infused rat; D, injured carotid artery from angiotensin II–infused rat, showing high levels of grains distributed diffusely throughout the thickness of the neointima and to a lesser extent the underlying media. Lumen is on top. Media is between the internal and external elastic laminae (arrowheads). Hematoxylin counterstain; bar=50 μm.

Figure 3.
Figure 3.

Immunohistochemical analysis of osteopontin protein levels in normal and injured carotid arteries of rats infused continuously with angiotensin II (250 ng/kg per minute) or Ringer's lactate 5 and 6 weeks after balloon injury to the left carotid artery. Carotid arteries were fixed in MethaCarnoy's and embedded, and 5-μm sections were stained for osteopontin with the polyclonal antiserum OP199 raised against rat smooth muscle osteopontin. A, Uninjured carotid artery media (between small arrowheads) from Ringer's-infused rat, showing no detectable immunostaining; B, uninjured carotid artery from angiotensin II–infused rat, showing medial cells positive for osteopontin (small arrow) as well as diffuse staining in the media; C, injured carotid artery from Ringer's-infused rat, showing no detectable immunostaining; D, injured carotid artery from angiotensin II–infused rat, showing osteopontin-positive cells situated predominantly near the luminal surface of the neointima (small arrows) but present also (large arrowheads) in the inner neointima and occasionally the underlying media. Lumen is on top. Neointima and media are separated by the internal elastic lamina (small arrowheads). Hematoxylin counterstain; bar=50 μm (A and B) and 20 μm (C and D).

Ang II–Stimulated Osteopontin Expression in Arteries With Intimal Thickening

We previously reported that osteopontin expression is increased transiently in smooth muscle cells over the first 3 weeks after balloon injury and that the increased expression is highly localized in the neointima.3 The present study confirms that basal levels of osteopontin expression are low in arteries at 6 weeks after injury (Fig 1⇑, top, lanes 3 and 5). Osteopontin mRNA in injured carotid arteries was markedly increased with Ang II infusion at 5 to 6 weeks after injury, as shown by Northern blot (Fig 1⇑, top, lanes 4 and 6) and in situ hybridization analyses (Fig 2C and 2D⇑⇑). Both of these methods indicated that Ang II stimulation of osteopontin gene expression was greater in the neointima than in the underlying media. In response to Ang II, the level of osteopontin mRNA in the injured artery media remained at 37% and 26% of levels in the neointima and uninjured artery media, respectively (Fig 1⇑). With Ang II, the in situ hybridization signal for osteopontin mRNA showed a marked increase distributed diffusely in the neointima and a marginal increase in the underlying media. The sense probe gave no localized signal in any of the injured arteries examined (not shown). Furthermore, Ang II infusion increased the expression of immunoreactive osteopontin protein above the detection level in the injured artery. Staining for osteopontin was mainly cell-associated and situated predominantly in the neointima. Osteopontin-positive cells were also seen occasionally in the media of injured arteries (Fig 3C and 3D⇑⇑).

Osteopontin is chemotactic for and produced by cells of the monocyte-macrophage lineage, for which the antigen ED-1 is a commonly used marker in rats.19 ED-1–positive cells were only rarely detected in either uninjured or injured carotid arteries, and the occurrence of these cells showed no association with the drug treatment (not shown). The few ED-1–positive cells seen in injured arteries were often located near the internal elastic lamina, deep in the neointimal lesion.

Ang II and Growth in the Arterial Wall

Ang II stimulated smooth muscle DNA replication in the media of the uninjured carotid artery (6.3-fold, Fig 4⇓; see BrdU labeling in Fig 5⇓) as well as in the media of the uninjured thoracic aorta (BrdU-labeling fraction of aortic smooth muscle cells in Ang II group, 24.2±5.7% versus Ringer's group, 2.5±0.5%; P<.05; n=6 rats per group). In carotid arteries with a previous injury, Ang II increased smooth muscle DNA replication in the neointima (6.9-fold) but failed to stimulate significant growth in the underlying media (Fig 4⇓).

Figure 4.
Figure 4.

Increased smooth muscle DNA synthesis in normal and injured carotid artery of rats infused continuously with angiotensin II (250 ng/kg per minute) 5 and 6 weeks after balloon injury to the left carotid artery. Control rats were infused with Ringer's lactate vehicle. All rats also received coinfusion of 5-bromo-2′-deoxyuridine via a separate pump to label replicating DNA in vivo. Values are mean±SE of the cumulative labeling fraction of smooth muscle cells in the arterial wall (n=6 rats per group), as described in “Methods.” *P<.05.

Figure 5.
Figure 5.

Nuclear incorporation of 5-bromo-2′-deoxyuridine (BrdU) in arteries of rats coinfused with BrdU (0.8 mg/kg per day) and either angiotensin II (250 ng/kg per minute) or Ringer's lactate 5 and 6 weeks after balloon injury to the left carotid artery. A, Uninjured artery from Ringer's-infused rat, showing BrdU-positive nucleus (stained in dark brown; small arrow) in the media (between small arrowheads); B, uninjured artery from angiotensin II–infused rat, showing several BrdU-positive nuclei (small arrow) in the media. BrdU-positive endothelial and adventitial cells were also frequently observed with angiotensin II infusion; C, injured artery from Ringer's-infused rat, showing BrdU-positive nuclei (small arrow) near the luminal surface of the neointima and none in the underlying media; D, injured artery from angiotensin II–infused rat, showing a marked increase in the number of BrdU-positive nuclei (small arrow) near the luminal surface of the neointima but none in the underlying media. Lumen is on top. Neointima and media are separated by the internal elastic lamina (small arrowheads). Hematoxylin counterstain; bar=20 μm.

Because in vitro studies have identified osteopontin as a putative growth-associated gene in smooth muscle cells,10 11 we sought to determine whether our present in vivo data supported this hypothesis. There was a highly significant (P<.001, R2=0.88) positive correlation in the media and neointima of arteries between the steady-state level of osteopontin mRNA (data from Fig 1⇑) and the frequency of BrdU-positive nuclei (data from Fig 4⇑).

In the media of uninjured carotid arteries, BrdU-positive cells (Fig 5⇑) and cells expressing osteopontin mRNA (Fig 2⇑) showed comparable distribution patterns. In the injured artery neointima, however, the patterns were dissimilar: BrdU-positive cells were highly localized to areas near the luminal surface of the neointima, in contrast to the in situ hybridization signal for osteopontin mRNA, which was more broadly distributed throughout the neointima.

Ang II infusion increased vascular mass in the uninjured carotid artery but induced no significant change in the media or neointima in the injured artery (Table⇓).

View this table:
Table 1.

Cross-sectional Area of Media and Neointima of Carotid Arteries of Rats Infused With Angiotensin II or Ringer's Lactate After Balloon Injury to the Left Carotid Artery

Arterial Osteopontin Expression in Catecholamine-Infused Rats

We examined arterial osteopontin expression in an Ang II–independent model of vascular growth and blood pressure regulation. In a previous study, we reported that a 2-week infusion of norepinephrine or phenylephrine in rats increased DNA replication by sixfold to sevenfold (BrdU labeling index) in the uninjured arterial wall while causing labile hypertension.15 Average systolic pressure during catecholamine infusion was not stably increased above 155 mm Hg versus control pressure values ranging between 125 and 135 mm Hg as measured by either tail-cuff plethysmography or radiotelemetry in conscious, unrestrained rats chronically catheterized via the abdominal aorta.15 In the present study, these catecholamine-infused rats overexpressed the osteopontin gene in the uninjured carotid artery media, as shown in Fig 6⇓. The ratio of osteopontin mRNA to 28S rRNA showed a marked increase with infusion of phenylephrine or norepinephrine (ratios of 4.8 and 4.3, respectively) versus the vehicle solution with or without ascorbate (ratios of 0.6 and 1.3, respectively). The positive correlation between arterial osteopontin expression and DNA replication remained highly significant after the data were pooled from the catecholamine and Ang II studies (P<.001, R2=0.82).

Figure 6.
Figure 6.

Top, Levels of osteopontin mRNA (size, 1.6 kb) in uninjured carotid artery media of rats infused continuously for 2 weeks with either Ringer's lactate (lane 1), the selective α1-adrenergic agonist phenylephrine (25 mg/kg per day, lane 2), norepinephrine (2.5 mg/kg per day, lane 3), or the drug vehicle Ringer's lactate containing 0.02% ascorbate (used as a preservative for catecholamines, lane 4). Each lane was loaded with 15 μg total RNA (pool of six rat arteries per group). Size of the observed bands was estimated on the basis of migration of the 18S and 28S rRNAs (revealed with methylene blue staining). Bottom, Methylene blue–stained filter shows 28S rRNA bands (size, 4 to 4.4 kb).

Discussion

To our knowledge, the present study is the first to document increased osteopontin expression in the arterial wall in response to vasopressor agents in vivo. Chronic infusion of Ang II to rats markedly stimulated osteopontin gene and protein expressions in smooth muscle cells in the media of uninjured arteries. In arteries with intimal thickening, however, Ang II induced a gradient of osteopontin expression across the vessel wall, as the highest mRNA levels for osteopontin were seen in the neointima. Tissue levels of osteopontin mRNA at the end of the treatment correlated with the frequency of cells having undergone DNA replication during the 2-week infusion period: uninjured artery media≈injured artery neointima>>injured artery media. In the neointima, however, osteopontin mRNA appeared more broadly distributed than newly replicated DNA, which was found predominantly at the luminal surface of the lesion. Ang II infusion caused severe hypertension. Osteopontin expression was also increased in phenylephrine-infused rats, an Ang II–independent model of increased vascular growth in which labile blood pressure elevations result in borderline hypertension.15

A major finding of the present study is the gradient of osteopontin expression induced by Ang II in arteries with intimal thickening: Osteopontin expression with Ang II was greater in the neointima than in the underlying media. This observation is supported by Northern blot quantification of osteopontin mRNA levels (Fig 1⇑) and is consistent with the histological data (Figs 2 and 3⇑⇑). Human atherosclerotic arteries also show a high localization of osteopontin expression to the intima as opposed to the media.3 5 6 The regulation of osteopontin expression in smooth muscle cells remains incompletely understood, but available data (including the present data) suggest that it may be phenotypically regulated.2 20 Neointimal smooth muscle cells express several phenotypic features reminiscent of developmentally immature smooth muscle cells,21 including the constitutive overexpression of osteopontin during culture in vitro compared with cells cultured from the uninjured adult artery media.2 22 23 In vivo, the induction of osteopontin is transient and downregulated completely within 4 weeks after balloon injury, suggesting that osteopontin gene and protein expressions are under strict control in the vascular wall.3 The present data with Ang II are the first to show the reinduction of vascular osteopontin in vivo beyond the initial phase of neointima formation. Again, reinduction occurred mainly in the neointima. Cells in the injured artery media also failed to respond mitogenically to Ang II at 5 to 6 weeks after injury, a finding that confirms and extends our previous observations that injured media cells do not replicate with Ang II infusion at 9 to 10 weeks or 6 months after injury.13 Moreover, cells cultured from the injured artery media at 9 weeks after injury show a reduced ability to proliferate in response to serum in vitro compared with cells cultured from the normal artery media.14 Taken together, these data suggest that smooth muscle cells in the injured artery media also may be phenotypically altered. Atrophy of the media underlying the plaque is a common feature of human atherosclerotic arteries.24 It is also possible that the pattern of osteopontin response in the injured arterial wall reflects alterations in angiotensin type 1 receptor levels and thus that osteopontin expression is a marker for Ang II activity. Against this possibility, however, we have recently observed (by quantitative autoradiography in frozen carotid artery sections) that levels of binding sites for Ang II were unchanged in the media at 10 weeks after injury and hence could not account for the lack of proliferative response to Ang II infusion compared with the uninjured media.13 Alternatively, the metabolism of Ang II in the injured arterial wall may be altered.

The correlation between the tissue levels of osteopontin mRNA and the frequency of replicating smooth muscle cells suggests a possible association between these two responses. Gadeau et al10 used late-passage smooth muscle cells isolated from the normal rat arterial media and reported that osteopontin overexpression was highly related to cell cycle progression, increasing during the late G1 phase through the G2/M phase in response to serum stimulation. Consistent with these data, previous studies showed that the time course of osteopontin induction after arterial injury follows closely the stimulation of DNA synthesis in the media and neointima.2 3 In contrast, Shanahan et al11 also cultured medial cells from uninjured rat arteries and reported that osteopontin expression was independent of the presence of serum and that osteopontin was constitutively elevated after dedifferentiation of the cells with culture in vitro. In the present studies, a greater number of neointimal cells responded to Ang II with increased osteopontin expression (as shown by in situ hybridization, Fig 2⇑) than with increased DNA replication (Fig 5⇑). Because the present BrdU labeling is cumulative over 14 days, whereas in situ hybridization reveals gene expression at the time of death only, an obligatory association would have resulted in BrdU-positive cells being either equal to or greater in number than osteopontin-positive cells. Thus, the present results do not support an obligatory association between osteopontin expression and DNA replication in smooth muscle cells in vivo. Consistent with this interpretation, we have recently shown that osteopontin mRNA is abundant4 and cell proliferation infrequent25 in pathological atherectomy specimens from both primary and restenotic coronary artery lesions. Alternatively, cells that express osteopontin mRNA without BrdU incorporation after the 2-week Ang II infusion may be in the late G1 phase, a transient phase of the cell cycle associated with osteopontin induction before DNA replication.10

Ang II may increase osteopontin expression in the arterial wall via a direct interaction with smooth muscle cells, as evidenced by the stimulating effect of the peptide in vitro.3 However, additional pathways are possibly active in vivo. For instance, α1-adrenoreceptor blockers partly reduce Ang II–induced DNA replication in uninjured arteries18 ; hence, it is possible that osteopontin induction during Ang II infusion may also depend in part on endogenous catecholamines acting via α1-adrenoreceptors. Consistent with this, osteopontin expression in the normal arterial wall was stimulated by infusion of either norepinephrine or the selective α1-agonist phenylephrine. The present study does not clarify whether blood pressure regulates arterial expression of osteopontin. However, the data do not suggest a close correlation between the severity of hypertension and levels of vascular osteopontin expression, as shown by comparison of the expression in two different models of blood pressure regulation, ie, Ang II–induced severe hypertension versus catecholamine-induced labile hypertension.

The accumulation of smooth muscle cells in the intima and the dystrophic calcification of intimal lesions causing a reduction in arterial compliance are two major events contributing to occlusive vascular diseases.21 26 Osteopontin expression correlates positively with the severity of atherosclerosis in human arteries, and accumulation of the protein is highly colocalized with calcium deposits in the atheroma.3 4 6 The Ang II pathway is implicated in the development of intimal lesions during experimental atherosclerosis27 28 29 30 31 and after balloon catheterization.32 33 34 35 36 37 Smooth muscle cells forming the neointima after vascular injury express higher levels of angiotensin-converting enzyme activity than cells in the underlying media or normal artery media.38 39 40 It is intriguing to speculate that Ang II may accelerate vascular lesion progression through the induction of osteopontin. Neointimal thickening after balloon injury to rat arteries is reduced by inhibitors of the Ang II pathway, at least in part via the inhibition of smooth muscle cell migration.41 We recently showed that osteopontin is chemotactic for vascular smooth muscle cells in vitro7 8 and that in vivo administration of a neutralizing antibody directed against osteopontin reduces neointima formation after balloon injury in the rat carotid artery mainly by the suppression of smooth muscle cell migration.42 Vascular osteopontin may also alter the structure and/or behavior of endothelial cells. For instance, osteopontin interaction with the αvβ3-integrin receptor increases endothelial cell adhesiveness in vitro.8 The αvβ3-receptor is also implicated in the regulation of vascular cell apoptosis (programmed cell death) during angiogenesis in vivo.43

Subcutaneous injection of purified osteopontin induces leukocyte chemotaxis in mice.44 We recently reported that the osteopontin overexpression in the inflammatory kidney of Ang II–infused rats precedes closely the appearance of leukocytes in the renal interstitium, suggesting that osteopontin may promote leukocyte recruitment and inflammation in the hypertensive kidney.45 In the present studies, we found no evidence that osteopontin induction in the carotid artery results in monocyte-macrophage infiltration. Two possibilities may explain this finding. First, the production of osteopontin may be too low to induce leukocyte chemotaxis from circulating blood. Few, if any, leukocytes can de detected in the neointima of injured rat arteries during the first 2 weeks after injury,46 ie, during the peak period of osteopontin expression. Osteopontin protein accumulation in the rat injured arterial wall is transient, suggesting a high metabolic or clearance rate for the protein in the rat.3 Second, Ang II may induce factors that counteract osteopontin actions on cell migration. Tenascin, for instance, is an extracellular matrix protein that is considered antiadhesive and is reportedly enriched in the neointima of rat arteries47 or in cultured smooth muscle cells stimulated with Ang II.48 49

In summary, we report that osteopontin expression was increased in arterial smooth muscle cells during chronic infusion of Ang II or catecholamine in rats, in correlation with the increase in DNA replication in the different smooth muscle layers studied although not in correlation with the severity of hypertension. Ang II induction of osteopontin was more important in the arterial neointima than in the underlying media, suggesting an association with smooth muscle cell phenotype. In the neointima, osteopontin expression was not restricted to cells showing DNA replication, suggesting a nonobligatory association between these two responses. Arterial smooth muscle cell expression of osteopontin may contribute to Ang II–dependent intimal lesion progression and to vascular remodeling events associated with renovascular diseases or hyperadrenergic disorders.

Acknowledgments

This work was supported in part by a Medical Research Council of Canada Fellowship award to D.D. at the Department of Pathology, University of Washington–Seattle, and by grants from the National Institutes of Health (HL-26405, HL-42270, DK-47659, HL-18645, and HL-03174). Special thanks are extended to Patti Polinski, Marie-France Ross, Hillel Schwartz, and Foon Tsui for assistance in surgery and histology and to Chris Covin for assistance in Northern blot analyses by PhosphorImager.

  • Received December 8, 1995.
  • Revision received February 15, 1996.
  • Revision received May 13, 1996.

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  • Angiotensin II Induction of Osteopontin Expression and DNA Replication in Rat Arteries
    Denis deBlois, Donna M. Lombardi, Enming J. Su, Alexander W. Clowes, Stephen M. Schwartz and Cecilia M. Giachelli
    Hypertension. 1996;28:1055-1063, originally published December 1, 1996
    https://doi.org/10.1161/01.HYP.28.6.1055
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