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Objective: Our purpose was to study in vitro whether phenotypically-distinct interstitial cell clones from bovine aortic valve (BVIC) possess different calcifying potential in response to endotoxin (lipopolysaccharide [LPS]) and phosphate (Pi)., Methods and Results: Among various clones of BVIC obtained by limited dilution technique we selected 4 clones displaying different growth patterns and immunophenotypes. Uncloned and cloned cells were treated with combinations of LPS (100 ng/mL) and Pi (2.4 mmol/L). Uncloned BVIC showed increased alkaline phosphatase activity (ALP) after treatment with LPS, which resulted in calcification after addition of Pi. Among BVIC clones, only Clone 1 (fibroblast-like phenotype) showed a relevant increase in ALP after LPS treatment in parallel with prevention of smooth muscle (SM) alpha-actin accumulation. No effect was observed in clonal cells harboring a more stable SM cell-like profile (Clone 4). None of the isolated clones calcified but mineralization was induced in the presence of LPS plus Pi when Clone 1 was cocultured with Clone 4 or after seeding on type I collagen sponges., Conclusions: Endotoxin and phosphate can act as valve calcification promoters by targeting specific fibroblast-like interstitial valve cells that possess a unique procalcific potential.

The adventitial layer surrounding the blood vessels has long been exclusively considered a supporting tissue the main function of which is to provide adequate nourishment to the muscle layers of tunica media. Although functionally interconnected, the adventitial and medial layers are structurally interfaced at the external elastic lamina level, clearly distinguishable at the maturational phase of vascular morphogenesis. Over the last few years the "passive" role that the adventitia seemed to play in experimental and spontaneous vascular pathologies involving proliferation, migration, differentiation, and apoptosis of vascular smooth muscle cells (VSMCs) has been questioned. It has been demonstrated that fibroblasts from the adventitia display an important partnership with the resident medial VSMCs in terms of phenotypic conversion, proliferation, apoptotic, and migratory properties the result of which is neointima formation and vascular remodeling. This article is an attempt at reviewing the major themes and more recent findings dealing with the phenotypic conversion process that leads adventitial "passive" (static) fibroblasts to become "activated" (mobile) myofibroblasts. This event shows some facets in common with vascular morphogenesis, ie, the process of recruitment, incorporation, and phenotypic conversion of cells surrounding the primitive endothelial tube in the definitive vessel wall. We hypothesize that during the response to vascular injuries in the adult, "activation" of adventitial fibroblasts is, at least in part, reminiscent of a developmental program that also invests, although with distinct spatiotemporal features, medial VSMCs.

We asked whether balloon-injured neointima formation in the presence of high/low serum cholesterol (CT) levels might be affected by dietary supplementation with fish oil (FO). To test this hypothesis, we examined the differentiation, proliferation, or apoptosis profile of smooth muscle cell (SMC) and adventitial cell response to a mild injury induced via a Fogarty catheter in the carotid artery of adult rabbits that had been fed a standard chow or 0.5% CT-enriched diet starting 4 weeks before the lesion. One week before surgery, animals received FO supplementation. This regimen was continued for the following 3 weeks. The effect of FO on the early proliferative/migratory response of carotid SMCs was also examined in 2- and 7-day-injured normocholesterolemic rabbits. As controls, animals subjected to 3-week endothelial injury and animals kept on a 7-week CT diet were used. Carotid cryosections from the various animal groups were evaluated for morphometry (image analysis), differentiation (immunofluorescence with monoclonal antibodies specific for smooth muscle markers, ie, myosin isoforms, SM22, and fibronectin), proliferation (bromodeoxyuridine incorporation), and apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling). FO treatment significantly reduced the development of intimal thickening in normocholesterolemic rabbits but had no efficacy in the presence of relatively higher serum CT levels. At day 2 (adventitia) and day 7 (neointima, media, and adventitia), the proliferation index of SMCs in FO-treated injured rabbits was markedly lower than in untreated injured controls. Concomitantly with the antiproliferative effect, FO was able to decrease the size of 2 cell types involved in the cell growth response to endothelial injury, namely, the "fetal-type" medial SMC subpopulation and the fibroblast-derived adventitial myofibroblasts. Thus, in our experimental conditions, a low CT level is a permissive condition for FO to prevent neointima formation to a considerable extent. This event is attributable to the early postinjury effect of FO on SMC/adventitial cell proliferation/differentiation patterns.

During the "response-to-injury" process after a mechanical insult to the porcine coronary arteries, the adventitial cells acquire the structural characteristics of myofibroblasts before being incorporated into smooth muscle (SM) layer. We assessed whether the SM-specific SM22 protein can be used as a tracer of adventitial cell-myofibroblast differentiation in the mild balloon injury of rabbit carotid artery. To achieve this goal, we used 2 monoclonal anti-SM22 antibodies (E-11 and 1-B8) and a molecular probe for the SM22alpha mRNA isoform in immunocytochemical and in situ hybridization experiments. The differentiation profile and the migratory and proliferative ability of activated adventitial cells were evaluated by a panel of antibodies to some SM and nonmuscle antigens and pulse- and end-labeling with bromo-deoxyuridine, respectively. In adventitial cells, SM22 antigenicity and SM22alpha mRNA were detectable at days 2 and 4 and, to a lesser extent, at days 7 and 21 after injury, particularly near the adventitia-media interface and mostly colocalizing with bromo-deoxyuridine-positive cells. The pulse-labeling experiments showed that the large majority of these cells penetrated the outermost layer of the tunica media without migrating to the subendothelial region. The phenotypic features of activated migrating and nonmigrating adventitial cells resembled those of vimentin-actin myofibroblast subtype and fetal-type SM cells. These findings indicate that a direct exposure of adventitia to the lumen is not required for phenotypic changes and proliferation/migration of these cells. After comparison of the SM22 expression in arterial vessels during early stages of development, we hypothesize that in the injured carotid artery the mural incorporation of adventitial cells and the spatiotemporal activation of SM22 expression are reminiscent of the vascular morphogenetic process and suggest the existence of a stem cell-like reservoir in adventitia. The early adventitial upregulation of SM22 expression in the injured vessel might be related to a multistep transition process in which nonmuscle cells are converted to myofibroblasts and, possibly, to SM cells.

Plasma lipoprotein(a) [Lp(a)] levels are largely genetically determined by sequences linked to the gene encoding apolipoprotein(a) [apo(a)], the distinct protein component of Lp(a). Apo(a) is highly polymorphic in length due to variation in the numbers of a sequence encoding the apo(a) kringle 4 domain, and plasma levels of Lp(a) are inversely correlated with apo(a) size. In 2 racially homogeneous Bantu populations from Tanzania differing in their dietary habits, we found that median plasma levels of Lp(a) were 48% lower in those living on a fish diet than in those living on a vegetarian diet. Considering the relationship between apo(a) size and Lp(a) plasma concentration, we have extensively evaluated apo(a) isoform distribution in the 2 populations to determine the impact of apo(a) size in the determination of Lp(a) values. The majority of individuals (82% of the fishermen and 80% of the vegetarians) had 2 expressed apo(a) alleles. Additionally, the fishermen had a high frequency of large apo(a) isoforms, whereas a higher frequency of small isoforms was found in the vegetarians. When subjects from the 2 groups were matched for apo(a) phenotype, the median Lp(a) value was 40% lower in Bantus on the fish diet than in those on the vegetarian diet. A significant inverse relationship was also found between plasma n-3 polyunsaturated fatty acids and Lp(a) levels (r=-0.24, P=0.01). The results of this study are consistent with the concept that a diet rich in n-3 polyunsaturated fatty acids, and not genetic differences, is responsible for the lower plasma levels of Lp(a) in the fish-eating Bantus and strongly suggest that a sustained fish-based diet is able to lower plasma levels of Lp(a).