The smooth muscle cell membrane during atherogenesis: a potential target for amlodipine in atheroprotection.

BACKGROUND: Atherosclerotic disease has been present in the human population apparently from the beginning of time. However, it has only been in the 20th century that improvements in the control of infectious diseases have allowed the average life span to increase to the point where atherosclerosis has been able to affect the general population. By the middle of the 20th century, atherosclerosis had reached epidemic levels, and it is currently pandemic and increasing worldwide. Despite its growing significance to health care, we still know relatively little about the cellular basis for plaque genesis in the vessel wall. Current thinking holds that atherosclerosis is caused by an unchecked chronic inflammatory process involving the cells of the arterial wall and their interaction with LDL and various inflammatory cells. Considerable evidence suggests that the principal insults underlying atherogenesis are serum dyslipidemias and oxidative stress mediated primarily by oxidized LDL. However, just how these insults alter the cell biology of vascular cells and lead to the atherosclerotic phenotype is still under intense investigation. Moreover, recent clinical trials have provided evidence that certain classes of drugs, including newer calcium channel blockers (CCBs), can remodel the arterial smooth muscle cell (SMC) membrane and inhibit the progression of atherosclerotic disease. METHODS: This review summarizes our current thinking on atherogenesis in the arterial SMC and considers recent developments regarding alterations in the SMC membrane during the very early period of atherogenesis. We also discuss how certain CCBs might operate to produce atheroprotection. RESULTS: The SMC membrane becomes enriched in unesterified cholesterol soon after the development of serum hypercholesterolemia. With excess membrane cholesterol, the membrane becomes thicker and develops distinct cholesterol domains. These alterations in the membrane increase the permeability of SMC to calcium and induce a variety of alterations in SMC function that contribute to cellular atherogenic processes during plaque genesis. Amlodipine, a third-generation CCB, markedly inhibits the progression of lesions. The explanation of this novel action may lie in the effects of this drug on various potential cellular targets. CONCLUSIONS: Evidence is accumulating that excess membrane cholesterol may contribute to the cellular defects responsible for the transformation of the SMC to the atherosclerotic phenotype. Amlodipine, which has membrane-remodeling properties, is emerging as an important atheroprotective drug.

Expression of the heterogenous nuclear ribonucleoprotein complex K protein and the prolyl-4-hydroxylase alpha-subunit in atherosclerotic arterial smooth muscle cells.

Smooth muscle cells (SMC) play a major role in the formation of atherosclerotic lesions found on major blood vessels. SMC proliferation, migration, and protein synthesis promote the progression of the early lesion, the fatty streak, into a complex myointimal fibrous plaque. To investigate altered gene expression in SMC during atherogenesis, we characterized differences between SMC from normal rabbits, rabbits fed a 2% cholesterol diet, and Watanabe Heritable Hyperlipidemic rabbits (WHHL). We detected and isolated a 501 bp cDNA fragment representing the A isoform of heterogenous nuclear ribonucleoprotein complex K (hnRNP-K) and a 281 bp cDNA fragment representing the prolyl-4-hydroxylase alpha-subunit (alphaPH) mRNAs. hn-RNP-K was upregulated in SMC from cholesterol-fed rabbits isolated in primary culture, as well as in SMC medial tissue from both the cholesterol-fed and WHHL rabbits. alphaPH was upregulated in SMC from the cholesterol-fed rabbits isolated in primary culture and in the tissue from WHHL rabbits. These data demonstrate genes consistent with increased proliferation and collagen production are upregulated in SMC during atherogenesis and may shed new light on gene expression changes and corresponding phenotype changes in SMC during atherogenesis.

Atheroprotection with amlodipine: cells to lesions and the PREVENT trial. Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial.

Oxidized lipid and calcium regulatory abnormalities appear to play important roles in early atherogenesis secondary to cholesterol enrichment of the cell membrane in endothelial and arterial smooth muscle cells (SMCs). However, the link between the two is poorly understood. The findings reviewed here demonstrate that amlodipine has membrane-modifying and antioxidant actions at the cell membrane level in addition to its classical calcium channel blocking properties. These multiple pharmacologic actions may explain the cellular mechanisms of the atheroprotective effects of amlodipine in spontaneous atherogenesis and in accelerated atherosclerotic syndromes. Recent animal model studies have demonstrated that amlodipine inhibits the progression of atherosclerotic lesions and protects against restenosis after angioplasty. Amlodipine inhibits the cholesterol-induced increase in calcium permeability in SMCs, and has been shown to repair abnormalities in SMC membrane structure. Recent data have also demonstrated that amlodipine has a marked antioxidant action in membrane bilayers enriched with polyunsaturated fatty acids. However, these findings have been in animal models only; the efficacy of amlodipine in atheroprotection in humans cannot be predicted. The PREVENT trial has therefore been launched to examine the atheroprotective potential of amlodipine in spontaneous lesion development in humans with ischemic heart disease and in the prevention of restenosis after angioplasty.

Cholesterol, calcium and atherosclerosis: is there a role for calcium channel blockers in atheroprotection?

It is well known that the atherogenic dyslipidemias of either elevated serum LDL or reduced HDL levels correlate with the degree and severity of atherosclerosis. However, how this leads to atherogenesis is poorly understood. A role for cellular oxidative stress mediated by oxidized LDL has gained widespread acceptance, but this pathway is unlikely to be the sole atherogenic signal. Recent evidence obtained from arterial smooth muscle cells (SMC) and endothelial cells (EC) is consistent with another pathway that may explain, in part, the early alterations contributing to the initiation of cellular atherogenic modifications. This pathway involves enrichment of the cell plasma membrane with cholesterol. In SMC, in vitro (cell culture) and in vivo (cholesterol feeding) experiments demonstrate that cholesterol enrichment of the SMC membrane occurs rapidly and is associated with an increase in membrane bilayer width, calcium permeability, and cell proliferation. Removal of excess membrane cholesterol with human HDL restores these alterations, suggesting that this membrane structural 'defect' mediates these changes in cell function. In vitro, the increased calcium permeability is inhibitable by calcium channel blockers (CCBs), but in vivo, a calcium 'leak' pathway develops that is virtually uninhibitable. It is not surprising that the literature on the application of CCBs for atheroprotection is not wholly convincing. However, with the advent of the new third generation of CCBs, new hope arises. One of the first CCBs of this generation is amlodipine (Norvasc), a charged dihydropyridine that has a remarkable pharmacologic profile. First, it is markedly lipophilic allowing it to partition readily into cell membranes. Second, in the membrane it has the ability to re-order, or restore, the 'swollen' membrane bilayer back to normal in atherosclerotic SMC. Third, it has potent antioxidant properties. Fourth, it appears to inhibit the expression of a variety of genes implicated in atherogenesis. Fifth, it is a CCB. Amlodipine has demonstrated atheroprotection in both rabbit and subhuman primate models of this disease. We propose that cellular alterations induced by enrichment of the cell membrane with cholesterol, which appears to modulate SMC to the atherosclerotic phenotype, are inhibitable by amlodipine through a combination of its varied pharmacologic properties. The potential for atheroprotection with amlodipine is currently being investigated in a human trial (PREVENT trial) and the results of this trial will determine the relevance of the preclinical findings to humans.

Western blotting and isoform analysis of cathepsin D from normal and malignant human breast cell lines.

Cathepsin D from normal (Hs578Bst) and malignant (MCF7, MDA-MB-231) breast cell lines has been characterized with regard to its kinetic properties, activity levels, precursor and processed M(r) forms, and isoform composition. Normal cell cathepsin D appears to have a more neutral pH optimum (pH 3.5) than the cancer cell line (pH 3.0-3.2) and greater activity between pH values of 4.0 to 4.5. The two cancer cell lines have approximately 1.5 to 2.0-fold increased total acid protease activity and 2 to 3-fold increased pepstatin-inhibitable protease activity (i.e. cathepsin D) when compared to the normal breast cell line. Western blotting indicates that a major processed form of cathepsin D for all three cell lines occurs at 31 kDa. The cancer cell lines contain significant amounts of cathepsin D precursors of 47 and 42 kDa whereas the normal cell line contains little if any of these precursors. Isoelectric focusing indicates that the normal cell line contains approximately 50% of its total acid protease activity at pIs above 4 whereas the cancer cell lines contain 70-80% of their protease activity at such pIs. In addition, the cancer cell lines contain two to three major isoforms between pIs of 5.5 and 6.3 which were not present in the normal cell line. The isoforms from pI values of 5.5 to 7.3 for all three cell lines are 100% pepstatin-inhibitable. In addition, Western blot analysis indicates that these isoforms contain the processed 31 kDa form of cathepsin D. The combined results indicate that the two breast cancer cell lines are similar to biopsied malignant breast tissue in exhibiting altered acid protease isoform profiles with increased relative amounts of pepstatin-inhibitable and immunoreactive acid protease activity (cathepsin D) compared to normal breast tissue or cells.

The exon-intron organization of the human erythrocyte alpha-spectrin gene.

The human erythrocyte alpha-spectrin gene which spans 80 kbp has been cloned from human genomic DNA as overlapping lambda recombinants. The exon-intron junctions were identified and the exons mapped. The gene is encoded by 52 exons whose sizes range from 684 bp to the smallest of 18 bp. The donor and acceptor splice site sequences match the splice site consensus sequences, with the exception of one splice site where a donor sequence begins with -GC. The size and location of exons do not correlate with the 106-amino-acid repeat, except in three locations where the surrounding codons are conserved as well. The lack of correspondence between exons and 106-amino-acid repeat is interpreted to reflect the appearance of a spectrin-like gene from a minigene early in the evolution of eukaryotes. Since current evidence indicates that introns were present in genes before the divergence of prokaryotes and eukaryotes, it is possible that the original distribution of introns within the minigene has been lost by the random deletion of introns from the spectrin gene.

Antibody-affinity purification of novel alpha-L-fucosidase from mouse liver.

Previous studies have documented the presence of a novel alpha-L-fucosidase in mouse liver that contains unique basic isoelectric forms and that is antigenically similar to, but not identical with, human liver alpha-L-fucosidase [Laury-Kleintop, Damjanov & Alhadeff (1985) Biochem. J. 230, 75-82]. In the present investigation, mouse liver alpha-L-fucosidase was purified approx. 26,500-fold in 10% overall yield by antibody-affinity chromatography with the IgG fraction of goat anti-(human alpha-L-fucosidase) antibody coupled to Sepharose 4B. Native polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis indicated that the mouse fucosidase is highly purified if not homogeneous. Isoelectric focusing demonstrated that all enzymic forms found in crude mouse liver supernatant fluids were purified by the antibody-affinity procedure.

Isoelectric focusing of alpha-L-fucosidase from human embryonal carcinoma.

Isoelectric focusing profiles of alpha-L-fucosidase recovered from human embryonal carcinoma (EC) and seminoma were compared with those of mouse germ cell tumor-derived stem cells and some human somatic cell neoplasms in an attempt to determine whether EC cells could be used as a source of human basic isoelectric forms of the enzyme previously identified in normal and malignant mouse embryonic cells. alpha-L-Fucosidase activity in all human tumors was associated with isoelectric forms in the isoelectric point (pl) range between approximately 4.5 and 7, corresponding to the range seen in normal human tissues. The basic isoelectric forms (pl values, 7.5-9.5) that predominate in the embryonic isoelectric focusing pattern in mouse were not found in human neoplasms. The present data illustrate another difference between human and mouse EC and show that mouse EC cells are not a complete replica of equivalent human tumor cells.

Isoelectric forms of alpha-L-fucosidase in mouse teratocarcinoma-derived cell lines.

The alpha-L-fucosidase isoenzyme pattern of mouse teratocarcinoma-derived cell lines was analyzed by isoelectric focusing and compared with the pattern of a mammary carcinoma as an example of a malignant somatic cell line. In addition, these isoenzyme patterns were compared with those of normal fetal and adult mouse tissues from an earlier study. In the normal early fetal and placental tissues as well as in embryonal carcinoma and yolk sac carcinoma cells the alpha-L-fucosidase activity is predominantly associated with basic forms of the enzyme. This embryonic pattern of alpha-L-fucosidase is characterized by one to three isoelectric forms of the enzyme with pI values ranging from 7 to 9.5 accounting for more than two-thirds of the total activity. In contrast, the mammary carcinoma pattern resembles adult somatic tissues and primarily expresses acidic enzymatic forms (which comprise approximately 80% of total activity). The somatic cell malignancies arising in retransplantable teratocarcinomas show varying isoenzyme patterns. Thus, a malignant fibrous histiocytoma expresses predominantly basic forms of the enzyme, whereas a leiomyosarcoma expresses approximately equal amounts of acidic and basic forms of the enzyme resembling in this respect late fetal or immature neonatal tissues. These findings show that the embryonal carcinoma and yolk sac carcinoma cells of the mouse express the embryonic isoenzyme pattern of alpha-L-fucosidase in contrast to malignant cells originating in somatic tissue, like mammary carcinoma, which express the adult pattern. Malignancies arising in somatic tissues of teratocarcinomas may retain the embryonic alpha-L-fucosidase phenotype or show a phenotype corresponding to late fetal or neonatal tissues in normal ontogeny.

Characterization of mouse liver alpha-L-fucosidase. Demonstration of unusual basic isoelectric forms of the enzyme that appear to be developmentally regulated.

Mouse tissues contain unusual basic isoelectric forms of alpha-L-fucosidase (with approximate isoelectric points of 8.3 and 9.0) in addition to the usual acidic and neutral forms previously described in tissues of other species. These unusual forms are very prominent in placenta and foetal tissues and comprise approx, 50-80% of total activity up to 11 days of postnatal development. By 15 days of postnatal development, the basic forms are diminished in amount and comprise not more than 25% of total activity. Neuraminidase treatment of adult mouse liver alpha-L-fucosidase led to significantly decreased amounts of acidic forms and increased amounts of the basic forms, suggesting that these forms are chemically related at least in part by sialic acid residues. Comparative kinetic studies on mouse liver, human liver and mouse placental alpha-L-fucosidases indicated that they have the same Km (0.05-0.06 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside but different pH optima and thermostability properties. Mouse liver alpha-L-fucosidase has one pH optimum (5.5) and an acidic shoulder (centred around pH 4.0) compared with two distinct optima (4.3 and 6.8) for the human liver enzyme. Mouse placental alpha-L-fucosidase has a pH-activity curve comparable with that of the mouse liver enzyme except that the acidic shoulder is absent. Mouse liver alpha-L-fucosidase is considerably more thermolabile after preincubation at 50 degrees C than are the human liver and mouse placental enzymes, which gave similar thermodenaturation curves. Immunochemical studies indicated that mouse and human alpha-L-fucosidases are dissimilar antigenically but exhibit some cross-reactivity. The IgG fraction of antibody prepared in goat against human liver alpha-L-fucosidase was ineffective by itself in immunoprecipitating mouse liver alpha-L-fucosidase, but 63% and 72% of the mouse liver and placental enzymes respectively could be immunoprecipitated in the double-antibody experiments under conditions that immunoprecipitated 92% of the human liver enzyme.