Potential roles of osteopontin and αVβ3 integrin in the development of coronary artery restenosis after angioplasty

Angioplasty procedures are increasingly used to reestablish blood flow in blocked atherosclerotic coronary arteries. A serious complication of these procedures is reocclusion (restenosis), which occurs in 30–50% of patients. Migration of coronary artery smooth muscle cells (CASMCs) to the site of injury caused by angioplasty and subsequent proliferation are suggested mechanisms of reocclusion. Using both cultured human CASMCs and coronary atherectomy tissues, we studied the roles of osteopontin (OPN) and one of its receptors, αvβ3 integrin, in the pathogenesis of coronary restenosis. We also measured the plasma levels of OPN before and after angioplasty and determined the effect of exogenous OPN on CASMC migration, extracellular matrix invasion, and proliferation. We found that cultured CASMCs during log phase of growth and smooth muscle cell layer of the coronary atherosclerotic tissues of patients express both OPN mRNA and protein at a significantly elevated level compared with controls. Interestingly, whereas the baseline plasma OPN levels in control samples were virtually undetectable, those in patient plasma were remarkably high. We also found that interaction of OPN with αvβ3 integrin, expressed on CASMCs, causes migration, extracellular matrix invasion, and proliferation. These effects were abolished when OPN or αvβ3 integrin gene expression in CASMCs was inhibited by specific antisense S-oligonucleotide treatment or OPN-αvβ3 interaction was blocked by treatment of CASMCs with antibodies against OPN or αvβ3 integrin. Our results demonstrate that OPN and αvβ3 integrin play critical roles in regulating cellular functions deemed essential for restenosis. In addition, these results raise the possibility that transient inhibition of OPN gene expression or blocking of OPN-αvβ3 interaction may provide a therapeutic approach to preventing restenosis.

Integration of multiple instructive cues by neural crest stem cells reveals cell-intrinsic biases in relative growth factor responsiveness

Growth factors can influence lineage determination of neural crest stem cells (NCSCs) in an instructive manner, in vitro. Because NCSCs are likely exposed to multiple signals in vivo, these findings raise the question of how stem cells would integrate such combined influences. Bone morphogenetic protein 2 (BMP2) promotes neuronal differentiation and glial growth factor 2 (GGF2) promotes glial differentiation; if NCSCs are exposed to saturating concentrations of both factors, BMP2 appears dominant. By contrast, if the cells are exposed to saturating concentrations of both BMP2 and transforming growth factor β1 (which promotes smooth muscle differentiation), the two factors appear codominant. Sequential addition experiments indicate that NCSCs require 48–96 hrs in GGF2 before they commit to a glial fate, whereas the cells commit to a smooth muscle fate within 24 hr in transforming growth factor β1. The delayed response to GGF2 does not reflect a lack of functional receptors; however, because the growth factor induces rapid mitogen-activated protein kinase phosphorylation in naive cells. Furthermore, GGF2 can attenuate induction of the neurogenic transcription factor mammalian achaete-scute homolog 1, by low doses of BMP2. This short-term antineurogenic influence of GGF2 is not sufficient for glial lineage commitment, however. These data imply that NCSCs exhibit cell-intrinsic biases in the timing and relative dosage sensitivity of their responses to instructive factors that influence the outcome of lineage decisions in the presence of multiple factors. The relative delay in glial lineage commitment, moreover, apparently reflects successive short-term and longer-term actions of GGF2. Such a delay may help to explain why glia normally differentiate after neurons, in vivo.

Heteromultimeric CLC chloride channels with novel properties

The skeletal muscle chloride channel CLC-1 and the ubiquitous volume-activated chloride channel CLC-2 belong to a large gene family whose members often show overlapping expression patterns. CLC-1 and CLC-2 are coexpressed in skeletal and smooth muscle and in the heart. By coexpressing CLC-1 and CLC-2 in Xenopus oocytes, we now show the formation of novel CLC-1/CLC-2 heterooligomers that yield time-independent linear chloride currents with a chloride → bromide → iodide selectivity sequence. Formation of heterooligomeric CLC channels increases the number and possible functions of chloride channels.

Localization and activity of lysyl oxidase within nuclei of fibrogenic cells

Lysyl oxidase (EC 1.4.3.13) oxidizes peptidyl lysine to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent cross-linkages that insolubilize these extracellular proteins. Recent findings raise the possibility that this enzyme may also function intracellularly. The present study provides evidence by immunocytochemical confocal microscopy, Western blot analysis, enzyme assays, and chemical analyses for lysyl oxidase reaction products that this enzyme is present and active within rat vascular smooth muscle cell nuclei. Confocal microscopy indicates its presence within nuclei of 3T3 fibroblasts, as well.

Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current regulation and seizure activity in mice

Muscarinic acetylcholine receptors are members of the G protein-coupled receptor superfamily expressed in neurons, cardiomyocytes, smooth muscle, and a variety of epithelia. Five subtypes of muscarinic acetylcholine receptors have been discovered by molecular cloning, but their pharmacological similarities and frequent colocalization make it difficult to assign functional roles for individual subtypes in specific neuronal responses. We have used gene targeting by homologous recombination in embryonic stem cells to produce mice lacking the m1 receptor. These mice show no obvious behavioral or histological defects, and the m2, m3, and m4 receptors continue to be expressed in brain with no evidence of compensatory induction. However, the robust suppression of the M-current potassium channel activity evoked by muscarinic agonists in sympathetic ganglion neurons is completely lost in m1 mutant mice. In addition, both homozygous and heterozygous mutant mice are highly resistant to the seizures produced by systemic administration of the muscarinic agonist pilocarpine. Thus, the m1 receptor subtype mediates M current modulation in sympathetic neurons and induction of seizure activity in the pilocarpine model of epilepsy.

Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyΔC1225 and MyΔC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.

Phosphorylation of the rRNA transcription factor upstream binding factor promotes its association with TATA binding protein

rRNA synthesis by RNA polymerase I requires both the promoter selectivity factor 1, which is composed of TATA binding protein (TBP) and three TBP-associated factors, and the activator upstream binding factor (UBF). Whereas there is strong evidence implicating a role for phosphorylation of UBF in the control of growth-induced increases in rRNA transcription, the mechanism of this effect is not known. Results of immunoprecipitation studies with TBP antibodies showed increased recovery of phosphorylated UBF from growth-stimulated smooth muscle cells. Moreover, using an immobilized protein-binding assay, we found that phosphorylation of UBF in vivo in response to stimulation with different growth factors or in vitro with smooth muscle cell nuclear extract increased its binding to TBP. Finally, we demonstrated that UBF–TBP binding depended on the C-terminal ‘acidic tail’ of UBF that was hyperphosphorylated at multiple serine sites after growth factor stimulation. Results of these studies suggest that phosphorylation of UBF and subsequent binding to TBP represent a key regulatory step in control of growth-induced increases in rRNA synthesis.

Estrogen inhibits the vascular injury response in estrogen receptor β-deficient female mice

The protective effects of estrogen in the cardiovascular system result from both systemic effects and direct actions of the hormone on the vasculature. Two estrogen receptors have been identified, ERα and ERβ. We demonstrated previously that estrogen inhibits the response to vascular injury in both wild-type and ERα-deficient mice, and that ERβ is expressed in the blood vessels of each, suggesting a role for ERβ in the vascular protective effects of estrogen. In the present study, we examined the effect of estrogen administration on mouse carotid arterial injury in ERβ-deficient mice. Surprisingly, in ovariectomized female wild-type and ERβ knockout mice, 17β-estradiol markedly and equally inhibited the increase in vascular medial area and the proliferation of vascular smooth muscle cells after vascular injury. These data demonstrate that ERβ is not required for estrogen-mediated inhibition of the response to vascular injury, and suggest that either of the two known estrogen receptors is sufficient to protect against vascular injury, or that another unidentified estrogen receptor mediates the vascular protective effects of estrogen.

Neuronal calcium sparks and intracellular calcium “noise”

Intracellular calcium ions are involved in many forms of cellular function. To accommodate so many control functions, a complex spatiotemporal organization of calcium signaling has developed. In both excitable and nonexcitable cells, calcium signaling was found to fluctuate. Sudden localized increases in the intracellular calcium concentration—or calcium sparks—were found in heart, striated and smooth muscle, Xenopus Laevis oocytes, and HeLa and P12 cells. In the nervous system, intracellular calcium ions were found important in key processes such as transmitter release, repetitive firing, and gene expression. Hence, we examined whether calcium sparks also exist in neurons. Using confocal laser-scanning microscopy and fluorescent probes, we found that calcium sparks exist in two types of neuronal preparations: the presynaptic boutons of the lizard neuromuscular junction and rat hippocampal neurons in cell culture. Control experiments exclude the possibility that these calcium sparks originate from instrumental or biological artifacts. Calcium sparks seem to be just the tip of the iceberg of a more general phenomenon of intracellular calcium “noise.” We speculate that calcium sparks and calcium noise may be of key importance in calcium signaling in the nervous system.

Localization of neuropeptide Y Y1 receptors in cerebral blood vessels

The localization of neuropeptide Y (NPY) Y1 receptor (R) -like immunoreactivity (LI) has been studied in cerebral arteries and arterioles of the rat by immunohistochemistry using fluorescence, confocal, and electron microscopy. High levels of Y1-R-LI were observed in smooth muscle cells (SMCs) in the small arterioles of the pial arterial network, especially on the basal surface of the brain, and low levels in the major basal cerebral arteries. The levels of Y1-R-LI varied strongly between adjacent SMCs. Y1-R-LI was associated with small endocytosis vesicles, mainly on the outer surface of the SMCs, but also on their endothelial side and often laterally at the interface between two SMCs. NPY-immunoreactive (Ir) nerve fibers could not be detected in association with the Y1-R-rich small arterioles but only around arteries with low Y1-R levels. A dense network of central NPY-Ir nerve fibers in the superficial layers of the brain was lying close to the strongly Y1-R-Ir small arterioles. The results indicate that NPY has a profound effect on small arterioles of the brain acting on Y1-Rs, both on the peripheral and luminal side of the SMCs. However, the source of the endogenous ligand, NPY, remains unclear. NPY released from central neurons may play a role, in addition to blood-borne NPY.

Expression of lipocalin-type prostaglandin D synthase (β-trace) in human heart and its accumulation in the coronary circulation of angina patients

Lipocalin-type prostaglandin D synthase (L-PGDS) is localized in the central nervous system and male genital organs of various mammals and is secreted as β-trace into the closed compartment of these tissues separated from the systemic circulation. In this study, we found that the mRNA for the human enzyme was expressed most intensely in the heart among various tissues examined. In human autopsy specimens, the enzyme was localized immunocytochemically in myocardial cells, atrial endocardial cells, and a synthetic phenotype of smooth muscle cells in the arteriosclerotic intima, and accumulated in the atherosclerotic plaque of coronary arteries with severe stenosis. In patients with stable angina (75–99% stenosis), the plasma level of L-PGDS was significantly (P < 0.05) higher in the great cardiac vein (0.694 ± 0.054 μg/ml, n = 7) than in the coronary artery (0.545 ± 0.034 μg/ml), as determined by a sandwich enzyme immunoassay. However, the veno-arterial difference in the plasma L-PGDS concentration was not observed in normal subjects without stenosis. After a percutaneous transluminal coronary angioplasty was performed to compress the stenotic atherosclerotic plaques, the L-PGDS concentration in the cardiac vein decreased significantly (P < 0.05) to 0.610 ± 0.051 μg/ml at 20 min and reached the arterial level within 1 h. These findings suggest that L-PGDS is present in both endocardium and myocardium of normal subjects and the stenotic site of patients with stable angina and is secreted into the coronary circulation.

Loperamide: A positive modulator for store-operated calcium channels?

The depletion of inositol trisphosphate-sensitive intracellular pools of calcium causes activation of store-operated calcium (SOC) channels. Loperamide at 10–30 μM has no effect on intracellular calcium levels alone, but augments calcium levels in cultured cells when SOC channels have been activated. In HL-60 leukemic cells, the apparent positive modulatory effect of loperamide on SOC channels occurs when these channels have been activated after ATP, thapsigargin, or ionomycin-elicited depletion of calcium from intracellular storage sites. Loperamide has no effect when levels of intracellular calcium are elevated through a mechanism not involving SOC channels by using sphingosine. Loperamide caused augmentation of intracellular calcium levels after activation of SOC channels in NIH 3T3 fibroblasts, astrocytoma 1321N cells, smooth muscle DDT-MF2 cells, RBL-2H3 mast cells, and pituitary GH4C1 cells. Only in astrocytoma cells did loperamide cause an elevation in intracellular calcium in the absence of activation of SOC channels. The augmentation of intracellular calcium elicited by loperamide in cultured cells was dependent on extracellular calcium and was somewhat resistant to agents (SKF 96365, miconazole, clotrimazole, nitrendipine, and trifluoperazine) that in the absence of loperamide effectively blocked SOC channels. It appears that loperamide augments influx of calcium through activated SOC channels.

Primary Mesenchymal Cells Isolated from SPARC-null Mice Exhibit Altered Morphology and Rates of Proliferation

SPARC (secreted protein acidic and rich in cysteine)/BM 40/osteonectin is a matricellular protein shown to function as a counteradhesive factor that induces cell rounding and as an inhibitor of cell proliferation. These activities have been defined in cell culture, in which interpretation has been complicated by the presence of endogenous SPARC. We therefore sought to determine whether cell shape and proliferation would be affected by the absence of SPARC. Mesangial cells, fibroblasts, and aortic smooth muscle cells were isolated from SPARC-null and age-matched, wild-type mice. In contrast to wild-type cells, SPARC-null mesangial cells exhibited a flat morphology and an altered actin cytoskeleton. In addition, vinculin-containing focal adhesions were distributed over the center of SPARC-null cells, whereas in wild-type cells, the number of focal adhesions was reduced, and these structures were restricted largely to the cell periphery. Although the SPARC-null fibroblasts did not display overt differences in cell morphology, the cells responded to exogenous recombinant SPARC by rounding up in a manner similar to that of wild-type fibroblasts. Thus, the expression of endogenous SPARC is not required for the response of cells to SPARC. Additionally, SPARC-null mesangial cells, fibroblasts, and smooth muscle cells proliferated faster than their respective wild-type counterparts. Null cells also showed a greater sensitivity to the inhibition of cell cycle progression by the addition of recombinant SPARC. The increased proliferation rate of SPARC-null cells appeared to be mediated, at least in part, by an increase in the cell cycle regulatory protein cyclin A. We conclude that the expression of SPARC influences the cellular architecture of mesangial cells and that SPARC plays a role in the regulation of cell cycle in mesangial cells, fibroblasts, and smooth muscle cells.

Inhibition of RhoA Translocation and Calcium Sensitization by In Vivo ADP-Ribosylation with the Chimeric Toxin DC3B

Pretreatment of intact rabbit portal vein smooth muscle with the chimeric toxin DC3B (10−6 M, 48 h; Aullo et al., 1993; Boquet et al. 1995) ADP-ribosylated endogenous RhoA, including cytosolic RhoA complexed with rhoGDI, and inhibited the tonic phase of phenylephrine-induced contraction and the Ca2+-sensitization of force by phenylephrine, endothelin and guanosine triphosphate (GTP)γS, but did not inhibit Ca2+-sensitization by phorbol dibutyrate. DC3B also inhibited GTPγS-induced translocation of cytosolic RhoA (Gong et al., 1997a) to the membrane fraction. In DC3B-treated muscles the small fraction of membrane-associated RhoA could be immunoprecipitated, even after exposure to GTPγS, which prevents immunoprecipitation of non-ADP–ribosylated RhoA. Dissociation of cytosolic RhoA–rhoGDI complexes with SDS restored the immunoprecipitability and ADP ribosylatability of RhoA, indicating that both the ADP-ribosylation site (Asn 41) and RhoA insert loop (Wei et al., 1997) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that RhoA plays a significant role in G-protein-, but not protein kinase C-mediated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo the Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.

Polycystin 1 is required for the structural integrity of blood vessels

Autosomal dominant polycystic kidney disease (ADPKD), often caused by mutations in the PKD1 gene, is associated with life-threatening vascular abnormalities that are commonly attributed to the frequent occurrence of hypertension. A previously reported targeted mutation of the mouse homologue of PKD1 was not associated with vascular fragility, leading to the suggestion that the vascular lesion may be of a secondary nature. Here we demonstrate a primary role of PKD1 mutations in vascular fragility. Mouse embryos homozygous for the mutant allele (Pkd1L) exhibit s.c. edema, vascular leaks, and rupture of blood vessels, culminating in embryonic lethality at embryonic day 15.5. Kidney and pancreatic ductal cysts are present. The Pkd1-encoded protein, mouse polycystin 1, was detected in normal endothelium and the surrounding vascular smooth muscle cells. These data reveal a requisite role for polycystin 1 in maintaining the structural integrity of the vasculature as well as epithelium and suggest that the nature of the PKD1 mutation contributes to the phenotypic variance in ADPKD.