Adult Rat Aorta Research Articles

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Distribution of α1-adrenoceptor subtype proteins in different tissues of neonatal and adult rats

Distribution of alpha(1)-adrenoceptor (alpha(1)AR) subtype (alpha(1A), alpha(1B), alpha(1D)) proteins in brain, heart, kidney, and liver of 1-week-old rats and in brain, heart, aorta, kidney, liver, vas deferens, prostate, and adrenal glands of adult rats was investigated by Western analysis, using receptor subtype specific polyclonal antibodies. High levels of immunoreactive alpha(1A)AR and alpha(1D)AR in brain and heart and of alpha(1B)AR in liver and heart of neonatal rats were detected. In adult rat tissues, the abundance of alpha(1A)AR protein was most marked in the brain, intermediate in heart, aorta, liver, vas deferens, and adrenals, and minimal in the kidney and prostate; relative to other tissues, the expression of alpha(1B)AR was higher in brain and heart and that of alpha(1D)AR in brain. All the three receptor subtypes increased with age in the brain cortex, whereas the abundance of alpha(1B)AR increased in the heart but decreased in the liver; alpha(1A)AR and alpha(1D)AR in liver, kidney, and heart were not affected by age. It is concluded that alpha(1)AR subtypes are widely expressed in different neonatal and adult rat tissues.

Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development.

Hepatoma-derived growth factor (HDGF) is the first member identified of a new family of secreted heparin-binding growth factors highly expressed in the fetal aorta. The biologic role of HDGF in vascular growth is unknown. Here, we demonstrate that HDGF mRNA is expressed in smooth muscle cells (SMCs), most prominently in proliferating SMCs, 8-24 hours after serum stimulation. Exogenous HDGF and endogenous overexpression of HDGF stimulated a significant increase in SMC number and DNA synthesis. Rat aortic SMCs transfected with a hemagglutinin-epitope-tagged rat HDGF cDNA contain HA-HDGF in their nuclei during S-phase. We also detected native HDGF in nuclei of cultured SMCs, of SMCs and endothelial cells from 19-day fetal (but not in the adult) rat aorta, of SMCs proximal to abdominal aortic constriction in adult rats, and of SMCs in the neointima formed after endothelial denudation of the rat common carotid artery. Moreover, HDGF colocalizes with the proliferating cell nuclear antigen (PCNA) in SMCs in human atherosclerotic carotid arteries, suggesting that HDGF helps regulate SMC growth during development and in response to vascular injury.

Mechanisms of stiffening and strengthening in media-equivalents fabricated using glycation.

We have recently reported that glycation can be exploited to increase the circumferential tensile stiffness and ultimate tensile strength of media-equivalents (MEs) and increase their resistance to collagenolytic degradation, all without loss of cell viability (Girton et al., 1999). The glycated MEs were fabricated by entrapping high passage adult rat aorta SMCs in collagen gel made from pepsin-digested bovine dermal collagen, and incubated for up to 10 weeks in complete medium with 30 mM ribose added. We report here on experiments showing that ME compaction due to traction exerted by the SMCs with consequent alignment of collagen fibrils was necessary to realize the glycation-mediated stiffening and strengthening, but that synthesis of extracellular matrix constituents by these cells likely contributed little, even when 50 micrograms/ml ascorbate was added to the medium. These glycated MEs exhibited a compliance similar to arteries, but possessed less tensile strength and much less burst strength. MEs fabricated with low rather than high passage adult rat aorta SMCs possessed almost ten times greater tensile strength, suggesting that alternative SMCs sources and biopolymer gels may yield sufficient strength by compositional remodeling prior to implantation in addition to the structural remodeling (i.e., circumferential alignment) already obtained.

Distribution of α<sub>1</sub>-adrenoceptor subtype proteins in different tissues of neonatal and adult rats

Distribution of alpha(1)-adrenoceptor (alpha(1)AR) subtype (alpha(1A), alpha(1B), alpha(1D)) proteins in brain, heart, kidney, and liver of 1-week-old rats and in brain, heart, aorta, kidney, liver, vas deferens, prostate, and adrenal glands of adult rats was investigated by Western analysis, using receptor subtype specific polyclonal antibodies. High levels of immunoreactive alpha(1A)AR and alpha(1D)AR in brain and heart and of alpha(1B)AR in liver and heart of neonatal rats were detected. In adult rat tissues, the abundance of alpha(1A)AR protein was most marked in the brain, intermediate in heart, aorta, liver, vas deferens, and adrenals, and minimal in the kidney and prostate; relative to other tissues, the expression of alpha(1B)AR was higher in brain and heart and that of alpha(1D)AR in brain. All the three receptor subtypes increased with age in the brain cortex, whereas the abundance of alpha(1B)AR increased in the heart but decreased in the liver; alpha(1A)AR and alpha(1D)AR in liver, kidney, and heart were not affected by age. It is concluded that alpha(1)AR subtypes are widely expressed in different neonatal and adult rat tissues.

Aortic Smooth Muscle Cells Interact with Tenascin-C through Its Fibrinogen-like Domain

The extracellular matrix protein tenascin-C is a multidomain protein that regulates cell adhesion. We used two different smooth muscle cell subtypes derived from adult and newborn rat aorta to investigate the interaction of tenascin-C or its various domains with these cells using an adhesion assay. Newborn cells were three times more adherent to tenascin-C than adult cells. Tenascin C-adhering cells remained round, whereas they spread rapidly on a fibronectin substrate. Adhesion assays showed the interaction between tenascin-C and newborn cells to be predominantly RGD-independent. Mg2+ increased newborn cell adhesion to tenascin-C in a concentration-dependent manner, whereas Ca2+ had no effect. To analyze the structure-function relationships of different domains of tenascin-C, we used recombinant full-length fibronectin-like and fibrinogen-like domains and various subdomains corresponding to the alternatively spliced regions of tenascin-C. The cells adhered to the fibrinogen-like domain but not to the fibronectin-like domain or its subdomains. As with the intact tenascin-C molecule, adherent cells remained round, and the Mg2+, but not Ca2+, promoted this interaction. The interaction of cells with the fibrinogen-like region was further mapped to a 30-amino acid peptide located near the carboxyl-terminal part of the tenascin-C molecule. The same 30-amino acid peptide was active in promoting cell migration. Our results provide a basis for understanding the mechanism of interaction of tenascin-C with smooth muscle cells and a framework for isolating membrane binding sites that mediate the cellular responses to this molecule.

Overexpression of P2Y2 purinoceptor in intimal lesions of the rat aorta.

Extracellular nucleotides, particularly ATP, are involved in the modulation of arterial vasomotricity via P2 purinoceptors present on smooth muscle and endothelial cells. These nucleotides could also be implicated in the smooth muscle cell hyperplasia observed in intimal lesions. In this study, we tried to define the potential role of the P2Y2 (P2u) purinoceptor by studying its expression in normal and balloon-injured rat aortas. The cloning of a rat P2Y2 cDNA from a rat smooth muscle cell cDNA library made it possible to study P2Y2 expression both by Northern blot and in situ hybridization. Northern blot experiments indicated that P2Y2 mRNA was present in rat medial aortic smooth muscle and in cultured rat aortic smooth muscle cells. In situ hybridization indicated that P2Y2 mRNA was present in endothelial cells of the intima and in some smooth muscle cells scattered throughout the media of adult rat aortas, while almost all medial smooth muscle cells of rat embryo aorta expressed this receptor. In contrast with adult aortic media, the majority of neointimal smooth muscle cells found in aortic intimal lesions either 8 or 20 days after balloon injury were positive for P2Y2 mRNA. Moreover, a subpopulation of neointimal cells localized at the luminal surface could be identified by a higher P2Y2 expression than the underlying neointimal smooth muscle cells. These data showing a strong expression of the P2Y2 purinoceptor in the neointima of injured arteries suggest that extracellular nucleotides may be involved, via this receptor, in the intimal hyperplasia and/or chronic constriction observed at the lesion site, and consequently in the restenotic process.

Alpha1D-adrenergic receptors and mitogen-activated protein kinase mediate increased protein synthesis by arterial smooth muscle.

Catecholamines may influence vascular smooth muscle cell (SMC) growth and vascular hypertrophic diseases. We previously demonstrated that stimulation of alpha1-adrenoceptors (AR) causes hypertrophy of vascular SMCs in vitro and in situ. Here, we used adult rat aorta SMCs that express alpha1D- and alpha1B-ARs (but not alpha1A-ARs) in vitro to examine the mechanisms and alpha1-AR subtypes involved. Norepinephrine (NE) increased protein synthesis and content in a time- and dose-dependent manner. To identify the responsible alpha1-AR subtype, we first documented the selectivity of two alpha1-AR subtype antagonists, BMY 7378 (alpha1D-AR antagonist) and chloroethylclonidine (CEC; alpha1B-AR antagonist), using Rat-1 fibroblasts stably transfected with the three different rodent alpha1-AR cDNAs. NE dose-dependently increased protein synthesis in each cell line. In alpha1D fibroblasts, BMY 7378 inhibited growth and protected alpha1D-ARs from CEC alkylation while having little blocking or protecting effect on the growth induced by stimulation of fibroblasts that express alpha1A- or alpha1B-ARs. In rat aorta SMCs, pretreatment with CEC in the presence of BMY 7378 to protect alpha1D-ARs had no effect on NE-induced protein synthesis. BMY 7378 inhibited the SMC growth response with a pKb of 8.4. NE caused rapid and transient p42-p44 mitogen-activated protein kinase (MAPK) activation that was alpha1D-AR dependent. Furthermore, NE caused tyrosine phosphorylation of multiple cellular proteins, phosphorylation of Raf-1, and stimulation of c-fos mRNA expression in aorta SMCs. The selective MAPK kinase inhibitor PD 98059 inhibited NE-induced protein synthesis and MAPK activation with IC50 values of 2.3 and 1.6 microM, respectively. These data demonstrate that SMC growth induced by NE is mediated by alpha1D-ARs that couple to activation of the MAPK cascade.

Lipopolysaccharide-responsive protein kinase C isotypes in the adult rat aorta.

Previous studies have shown that protein kinase C (PKC) activity increases in cardiovascular tissue exposed to lipopolysaccharide (LPS). The objective of these experiments was to identify the PKC isotypes that respond to LPS treatment in the adult rat aorta. We found that PKC alpha, -delta, -epsilon, and -zeta isotypes are present in endothelium-intact aortas. The PKC alpha and -epsilon isotypes show two- to threefold increases in abundance after 3 h treatment with 100 ng/mL LPS, while PKC delta and -zeta levels do not increase. In contrast, mRNA for all of the PKC isotypes increased 3.5 to 12-fold during LPS treatment. Both PKC isotype and mRNA levels gradually diminished during 20 h of continuous LPS exposure. Concurrent treatment of the vessels with LPS plus 50 microM cycloheximide caused PKC alpha, -epsilon, and -zeta, but not -delta, isotypes to rapidly decrease in abundance while blunting the increase in PKC isotype mRNA. The major source for all of the PKC isotypes in the vessel is the vascular smooth muscle cells. These results indicate that LPS treatment induces time-dependent increases in PKC isotype mRNA expression and isotype-specific PKC activation and synthesis in vascular tissue.

Effect of age on the vasodilatory action of elastin peptides

We have recently shown, on young adult rat aorta rings, that elastin peptides induce a dose and endothelium-dependent vasodilation mediated by the 67 kDa subunit of the high affinity elastin–laminin receptor and, at least in part, by EDRF (NO) [1]. Here we have studied the effects of elastin peptides at circulating concentrations and below, on noradrenaline-contracted rat aortic rings, as a function of age. First, we have observed that, unlike 2-month-old (2M), 4–6-month-old (4M) and 12-month-old (12M) rat aorta rings, 30-month-old (30M) rat aorta rings were unable to maintain their contraction in long lasting experiments. Secondly, elastin peptides at physiological circulating concentrations (10 −6–10 −3 mg/ml) induce a dose-dependent vasodilation on 4M rings. By contrast, only higher elastin peptide concentrations (10 −3 mg/ml) were effective on 12M rings, whereas rings from both younger (2M) and older animals (30M) did not respond to elastin peptides. Finally, using lactose and laminin as inhibitors, we have demonstrated that elastin peptide-induced vasodilation on 4M and 12M rings is mediated by the 67 kDa subunit of the elastin–laminin receptor. These experiments suggest that the functional availability of the 67 kDa subunit of the elastin–laminin receptor changes with age. It could be hypothesized that in young animals (0–2M) the reusable shuttle role recently demonstrated for the 67 kDa receptor subunit during elastic fiber formation [2]leads to a major decrease in its availability for signal transduction. On the contrary, in adult animals (4–12M), when developmental elastogenesis is completed, this subunit is essential for extracellular signal transduction [3]. Inefficiency of this receptor in old animals (30M) can be attributed to its uncoupling from its transduction pathway, as previously shown on human cells [4]. Finally, the age-dependent variations of circulating elastin peptide concentration and elastin–laminin receptor responsiveness to elastin peptides are two independent parameters which could influence the vascular tension regulation.

Alpha 1D-adrenoceptors do not contribute to inotropic responses of neonatal rat myocardium.

This study investigated the role of alpha 1D-adrenoceptors (ARs) in the inotropic responses of neonatal (7-day-old) rat myocardium by using the relatively selective antagonist BMY 7378. The positive inotropic effects of phenylephrine on right ventricular strips of neonatal rats were not inhibited by BMY 7378 up to a concentration of 300 nM but potently antagonized by the alpha 1AAR antagonist 5-methylurapidil (5-MU) (pKB, 8.73 +/- 0.12; n = 6). BMY 7378 was approximately 1,000-fold less potent (pKi, 6.63 +/- 0.7; n = 5) in inhibiting the binding of [3H]prazosin to right ventricular membrane preparations of neonatal rats than was 5-MU (pKi, 9.04 +/- 0.54; n = 5). Neonatal rat cerebral cortex and adult rat aorta were used as additional controls. BMY 7378 was a weak inhibitor of [3H]prazosin binding to neonatal cortex (pKi, 6.8 +/- 0.04; n = 3) but highly potent in inhibiting the binding to adult rat aortic membrane preparations (pKi for high-affinity binding, 9.70 +/- 0.30; n = 4); BMY 7378 potently antagonized the effects of phenyleprine on adult rat aortic strips (pKB, 9.05 +/- 0.11; n = 5). It is concluded that alpha 1DARs do not play a significant role in alpha 1AR-mediated inotropic responses of neonatal rat myocardium.

Long-term guanethidine sympathectomy suppresses flow-induced release of ATP and endothelin from endothelial cells isolated from adult rat aorta.

Chronic rather than acute changes in the autonomic innervation of the vasculature are a feature of ageing and several cardiovascular disorders. To investigate the long-term influence of perivascular innervation on the vascular endothelium, the release of vasoactive substances which have been localized in endothelial cells, namely ATP, endothelin, substance P and vasopressin, was monitored from cells isolated from adult rat thoracic aorta following neonatal guanethidine sympathectomy. The endothelial cells were initially perfused at 0.5 ml/min and exposed to two periods of increased flow at 3.0 ml/min. Cells isolated from control rats released significantly more ATP on both occasions of switching from the lower to higher flow rate and significantly more endothelin on the second exposure to the higher flow rate. In contrast, endothelial cells isolated from sympathectomised rats showed no increased release of either ATP or endothelin with increase flow, although the release of endothelin at the initial flow rate of 0.5 ml/min was higher than in the controls. Substance P and vasopressin levels in the perfusate were the same in controls and after sympathectomy. In summary, long-term sympathectomy suppresses increased flow-induced release of selected vasoactive substances from the endothelium, thus shear-stress-induced changes in local blood flow may be impaired when there are chronic disturbances in the autonomic innervation.

Heme oxygenase activity in the adult rat aorta and liver as measured by carbon monoxide formation.

Rat aorta was homogenized and the 13000 x g supernatant fraction was tested for heme oxygenase (HO) activity by using a sensitive gas chromatographic method to measure carbon monoxide (CO), one of the products of the HO reaction. The rate of NADPH-dependent CO formation, an index of HO activity, was 1.41 +/- 0.40 nmol CO.mg(-1)protein. h(-1) in the rat aorta supernatant fraction and 2.05 +/- 0.55 nmol CO.mg(-1) protein.h(-1) in the rat liver 13000 x g supernatant fraction, a tissue known to contain HO activity. Chromium mesoporphyrin (0.05 mM), an inhibitor of rat liver HO, significantly decreased HO activity by 26% in the aorta supernatant fraction and 50% in the liver supernatant fraction. On the basis of the results of this study, which demonstrated HO-catalyzed CO formation in aortic tissue, and previous observations that CO relaxes vascular smooth muscle, we suggest that a physiological role for CO in vascular smooth muscle relaxation should be further investigated.

Blood Pressure and Heat Shock Protein Expression in Response to Acute and Chronic Stress

We previously demonstrated that restraint and pharmacological agents that activate sympathetic nervous system activity induce expression of the 70-kD heat shock protein (HSP70) in major blood vessels. The magnitude and rapidity in which HSP70 is induced in the aorta suggest that it may play a salient role in the mechanical properties of vascular smooth muscle. Other investigators have reported that HSP70 inducibility is increased in genetically hypertensive animals. In this report, we have investigated the effects of acute and chronic (8-week) exposure to restraint and restraint in the presence of a randomized intermittent air jet on the development of hypertension and the induction of HSP70 in the aorta and adrenal glands of normotensive adult male Sprague-Dawley rats. Acute restraint or air jet resulted in a fivefold to sixfold increase in aortic HSP70 mRNA expression. Chronic exposure to restraint reduced the HSP70 response to acute restraint. In contrast, no adaptation of the HSP70 response to acute air jet was observed in aortas of chronically air jet-treated rats. In adrenal glands, HSP70 expression was reduced after chronic restraint and air jet, indicating that in this tissue, adaptation occurs to both stressors. There was no difference in HSP70 expression in unstressed rats that had been chronically exposed to restraint or air jet in either adrenal gland or aorta. A significant increase (P < .05) in systolic blood pressure developed in air jet-treated animals (120 +/- 3 mm Hg) but not in restrained rats (107 +/- 2 mm Hg) compared with unstressed controls (106 +/- 3 mm Hg).(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical stimuli induce vascular parathyroid hormone-related protein gene expression in vivo and in vitro

PTH-related peptide (PTHrP), the factor mediating the syndrome of humoral hypercalcemia of malignancy, is also expressed in smooth muscle cells (SMC) of the urinary bladder and uterus in response to mechanical distention and fetal occupancy, respectively. Vascular SMC also produce PTHrP, and its expression is induced by serum and vasoconstrictors, such as angiotensin-II. To determine whether mechanical distension affected vascular PTHrP gene expression, the abdominal aorta of adult male rats was balloon-distended, and aortae were collected at various times after the intervention. PTHrP mRNA was determined by competitive reverse transcriptase-polymerase chain reaction, using sequential dilutions of a cloned internally truncated PTHrP RNA fragment as standard. The molar concentration of PTHrP mRNA was obtained by extrapolating at a standard/wild-type band intensity ratio of 1:1. Aortic PTHrP mRNA was induced from a basal level of 19, to 22, 46, 36, 13, 12, 22, and 20 attamoles/mg total RNA 1, 2, 12, 24, and 48 h and 7 and 48 days after balloon distension, respectively. To determine whether mechanical events directly regulate vascular PTHrP gene expression, primary rat aortic SMC were plated and placed on a rocking device at 20 oscillations/min to create a gentle flowing motion of the culture medium. Rocking induced PTHrP mRNA of SMC exposed to either serum-free medium or 10% serum by 2.5-and 4.0-fold at 4 h, and 2.9- and 3.7-fold at 24 h, respectively. These effects were oscillation rate dependent, potentiated by angiotensin-II, and specific, as similar changes were not observed in alpha-actin mRNA content. Flow motion-induced PTHrP mRNA at 24 h was partially decreased by 10(-6) M colchicine (which inhibits microtubule assembly), but not by cytochalasin-E (which disrupts actin polymerization). As PTHrP is a known vasorelaxant, we propose that mechanical events induce the release of PTHrP by SMC, possibly to serve as a compliance factor or an agent for vascular remodeling.

Mechanical stimuli induce vascular parathyroid hormone-related protein gene expression in vivo and in vitro.

PTH-related peptide (PTHrP), the factor mediating the syndrome of humoral hypercalcemia of malignancy, is also expressed in smooth muscle cells (SMC) of the urinary bladder and uterus in response to mechanical distention and fetal occupancy, respectively. Vascular SMC also produce PTHrP, and its expression is induced by serum and vasoconstrictors, such as angiotensin-II. To determine whether mechanical distension affected vascular PTHrP gene expression, the abdominal aorta of adult male rats was balloon-distended, and aortae were collected at various times after the intervention. PTHrP mRNA was determined by competitive reverse transcriptase-polymerase chain reaction, using sequential dilutions of a cloned internally truncated PTHrP RNA fragment as standard. The molar concentration of PTHrP mRNA was obtained by extrapolating at a standard/wild-type band intensity ratio of 1:1. Aortic PTHrP mRNA was induced from a basal level of 19, to 22, 46, 36, 13, 12, 22, and 20 attamoles/mg total RNA 1, 2, 12, 24, and 48 h and 7 and 48 days after balloon distension, respectively. To determine whether mechanical events directly regulate vascular PTHrP gene expression, primary rat aortic SMC were plated and placed on a rocking device at 20 oscillations/min to create a gentle flowing motion of the culture medium. Rocking induced PTHrP mRNA of SMC exposed to either serum-free medium or 10% serum by 2.5-and 4.0-fold at 4 h, and 2.9- and 3.7-fold at 24 h, respectively. These effects were oscillation rate dependent, potentiated by angiotensin-II, and specific, as similar changes were not observed in alpha-actin mRNA content. Flow motion-induced PTHrP mRNA at 24 h was partially decreased by 10(-6) M colchicine (which inhibits microtubule assembly), but not by cytochalasin-E (which disrupts actin polymerization). As PTHrP is a known vasorelaxant, we propose that mechanical events induce the release of PTHrP by SMC, possibly to serve as a compliance factor or an agent for vascular remodeling.

Heparin decreases the rate of proliferation of rat vascular smooth muscle cells by releasing transforming growth factor beta-like activity from serum.

Various heparins have been reported to inhibit the proliferation of vascular smooth muscle cells (VSMCs). The effects of eight chemically distinct heparins on the cell cycle and differentiation of primary and passaged cultures of rat aortic VSMCs have been characterised and the mechanism of heparin action investigated. VSMCs from adult rat aorta were prepared by enzyme dispersion and stimulated to enter the cell cycle with 10% serum in the presence or absence of heparin. Progressions through S phase and M phase were measured by [3H]-thymidine incorporation and cell counting respectively. Flow cytometry was used to confirm the effects of heparin on VSMC cell cycle progression. The effect of heparin on VSMC differentiation was investigated by analysing smooth muscle specific myosin heavy chain content of the cells after heparin treatment. Eight heparins at concentrations between 5 micrograms.ml-1 and 100 micrograms.ml-1 partially inhibited VSMC proliferation (27% to 76% 96 hours after addition of heparin), but did not affect the entry of the cells into S phase. Flow cytometry confirmed that VSMC populations in the presence of heparin contained significantly (p < 0.005) more cells in the G2/M phase of the cell cycle than control populations. Heparin also blocked the dedifferentiation of primary cultures of VSMCs stimulated by serum. These effects of heparin were completely reversed by the presence of a neutralising antiserum to transforming growth factor beta (TGF beta) and heparin attached to agarose beads was as effective as free heparin as a growth inhibitor of VSMCs. Heparins of varying molecular weight and anticoagulant properties all partially inhibited VSMC proliferation predominantly by extending the G2/M phase of the cell cycle. Heparin also inhibited dedifferentiation of primary cultures of VSMCs. Heparin (< 100 micrograms.ml-1) acted extracellularly to release TGF beta from serum, which accounted for the effects of heparin on proliferation and differentiation.

Expression of follistatin, an activin-binding protein, in vascular smooth muscle cells and arteriosclerotic lesions.

Activin-A has a mitogenic effect on vascular smooth muscle cells (SMCs) and is produced by monocyte/macrophage lineage cells. Here, we studied the expression of follistatin, an activin-binding protein, in both A10 cells, a rat aortic SMC line, and vascular SMCs derived from adult rat aorta. Follistatin mRNA was detected in these cells by Northern blot analysis. Ligand blot and immunoblot analyses demonstrated that follistatin was produced in the conditioned medium at a higher level by A10 cells than by SMCs. Furthermore, immunostaining and in situ hybridization of the arteriosclerotic lesions showed that follistatin was highly expressed in the diseased artery, where abnormal proliferation of SMCs occurred. We suggest that follistatin is produced by vascular SMCs and is involved in the course of atherogenesis.

Osteopontin is elevated during neointima formation in rat arteries and is a novel component of human atherosclerotic plaques.

In an earlier report, we used differential cloning to identify genes that might be critical in controlling arterial neointima formation (Giachelli, C., N. Bae, D. Lombardi, M. Majesky, and S. Schwartz. 1991. Biochem. Biophys. Res. Commun. 177:867-873). In this study, we sequenced the complete cDNA and conclusively identified one of these genes, 2B7, as rat osteopontin. Using immunochemistry and in situ hybridization, we found that medial smooth muscle cells (SMC) in uninjured arteries contained very low levels of osteopontin protein and mRNA. Injury to either the adult rat aorta or carotid artery using a balloon catheter initiated a qualitatively similar time-dependent increase in both osteopontin protein and mRNA in arterial SMC. Expression was transient and highly localized to neointimal SMC during the proliferative and migratory phases of arterial injury, suggesting a possible role for osteopontin in these processes. In vitro, basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and angiotensin II (AII), all proteins implicated in the rat arterial injury response, elevated osteopontin expression in confluent vascular SMC. Finally, we found that osteopontin was a novel component of the human atherosclerotic plaque found most strikingly associated with calcified deposits. These data implicate osteopontin as a potentially important mediator of arterial neointima formation as well as dystrophic calcification that often accompanies this process.

Adult human aortic smooth muscle cells in culture produce active TGF-beta

Vascular smooth muscle cells (VSMC) from adult human aortas proliferated in culture in response to fetal calf serum (FCS) with a population doubling time of 70-85 h compared with 35 +/- 5 h for VSMC derived from adult rat aortas. Medium conditioned on cultures prepared from aortas from three different donors and mixed 1:1 with fresh Dulbecco's modified Eagle's medium plus 20% FCS [human conditioned medium (HCM)] reduced the rate of proliferation of rat VSMC by 46 +/- 6% (n = 3) after 48 h compared with cells in fresh medium. HCM did not reduce the proportion (> 65%) of rat VSMC that entered DNA synthesis but delayed entry into mitosis by at least 18 h. This effect was similar to previous observations of the action of transforming growth factor-beta (TGF-beta) on rat VSMC (G. K. Owens, A. A. Geisterfer, Y. W. Yang, and A. Komoriya. J. Cell Biol. 107: 771-780, 1988). A TGF-beta assay using DNA synthesis in mink lung epithelial cells confirmed that human, but not rat, VSMC in culture secrete active TGF-beta. Addition of a neutralizing antibody to TGF-beta to human VSMC in the presence of 20% FCS decreased the population doubling time from 74 +/- 3 to 46 +/- 6 h (n = 3). These observations demonstrate that the long population doubling time of human VSMC is due to the production of active TGF-beta and to an inhibitory autocrine loop.