A novel vascular smooth muscle chymase is upregulated in hypertensive rats.

While greater than 80% of angiotensin II (Ang II) formation in the human heart and greater than 60% in arteries appears to result from chymase activity, no cardiovascular cell-expressed chymase has been previously reported. We now describe the cloning of a full-length cDNA encoding a novel chymase from rat vascular smooth muscle cells. The cDNA encompasses 953 nucleotides, encodes 247 amino acids, and exhibits 74% and 80% homology in amino acid sequence to rat mast cell chymase I and II, respectively. Southern blot analysis indicates that the rat vascular chymase is encoded by a separate gene. This chymase was induced in hypertrophied rat pulmonary arteries, with 11-fold and 8-fold higher chymase mRNA levels in aortic and pulmonary artery smooth muscle cells from spontaneously hypertensive than in corresponding tissues from normotensive rats. We assayed the activity of the endogenous enzyme and of a recombinant, epitope-tagged chymase in transfected smooth muscle cells and showed that Ang II production from Ang I can be inhibited with chymostatin, but not EDTA or captopril. Spontaneously hypertensive rats show elevated chymase expression and increased chymostatin-inhibitable angiotensin-converting activity, suggesting a possible role for this novel enzyme in the pathophysiology of hypertension.

Increase in nuclear calcium in smooth muscle cells exposed to oxidized low density lipoprotein.

Vascular smooth muscle cells respond with an increase in intracellular Ca2+ within seconds after exposure to oxidized low density lipoprotein (oxLDL). This has been suggested to represent a signaling response that may have implications for gene expression. If so, oxLDL may induce increases in nuclear Ca2+ in smooth muscle cells in response to oxLDL. Aortic smooth muscle cells were exposed to 100 microg/ml oxLDL. Large, rapid increases in [Ca2+]i were observed using fluo-3 as an indicator dye to detect intracellular Ca2+ on the stage of a confocal microscope. This was also confirmed using ratiometric imaging of indo signals. These elevations appeared to be localized to the nuclear region of the cell. DNA staining of the cells confirmed its localization to the nuclear/perinuclear region of the cell. Our data demonstrate that oxLDL induces a nuclear localized elevation in Ca2+i that may have important implications for nuclear function.

Overexpression of SERCA2 Atpase in vascular smooth muscle cells treated with oxidized low density lipoprotein.

Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca2+(i) homeostasis and altered two Ca2+ release mechanisms in these cells (IP3 and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca2+ pump. One of the primary Ca2+ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP3 and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca2+ homeostasis in VSMC chronically exposed to atherogenic oxLDL.

Lesions in ryanodine channels in smooth muscle cells exposed to oxidized low density lipoprotein.

The purpose of the present investigation was to investigate the subcellular basis responsible for the loss of vasoreactivity in atherosclerotic vessels. We have chosen to focus on the potential of oxidized low density lipoprotein (oxLDL), an important atherogenic agent, to alter sarcoplasmic reticulum (SR) structure and function. Vascular smooth muscle cells (VSMCs) were exposed for 1 to 6 days to low concentrations of minimally oxidized LDL. ATP was used to probe SR function in VSMCs. ATP can increase [Ca(2+)](i) in control VSMCs because of a release of Ca(2+) from the SR. However, after chronic exposure to oxLDL, cells lose their ability to increase [Ca(2+)](i) in response to ATP. These cells also exhibit a depressed rise in [Ca(2+)](i) after exposure to ryanodine. These effects were associated with a decreased immunoreactivity for the ryanodine-sensitive Ca(2+)-release channels in the SR of oxLDL-treated cells. Immunohistochemical analysis of aortic sections obtained from rabbits fed a cholesterol-supplemented diet revealed a significant decrease in the immunoreactivity for ryanodine channels in the plaque and in the medial layer underlying the plaque. In summary, our data identify oxLDL as a component within the atherosclerotic milieu capable of inducing a decrease in smooth muscle ryanodine channel density. This alteration is associated with a significant defect in the ability of the SR within the smooth muscle cell to regulate Ca(2+). These lesions may contribute to the altered vasoreactivity exhibited by atherosclerotic vessels.

Oxidized low-density lipoprotein induces cytoskeletal disorganization in smooth muscle cells.

Vascular smooth muscle cells in atherosclerotic vessels proliferate and change from a contractile to a synthetic phenotype. To determine whether oxidized low-density lipoprotein (oxLDL) is involved in this transformation, we chronically incubated cultured smooth muscle cells with native and oxidized LDL. Western blot analysis detected a decrease in actin and myosin content in treated cells. This was dependent on the time and concentration of oxLDL employed. Confocal microscopic images of cells immunostained for smooth muscle-specific alpha-actin and myosin showed a normal, elongated alignment of myofilaments in cells after incubation with native LDL. Surprisingly, when the cells were treated with oxLDL, actin and myosin filaments underwent a striking process of disorganization and accumulation into ball-shaped aggregates. These changes were dependent on the duration and concentration of oxLDL employed. Our results demonstrate that oxLDL has the capacity to decrease the content of myofilaments in smooth muscle cells. The loss in myosin and actin protein may be associated with an unusual formation of large cellular aggregates that appear to be in the process of being expelled from the cell.

Chronic exposure of smooth muscle cells to minimally oxidized LDL results in depressed inositol 1,4,5-trisphosphate receptor density and Ca(2+) transients.

Oxidized LDL (oxLDL) (0.1 mg/mL) increased [Ca(2+)](i) in vascular smooth muscle cells (VSMCs) within 5 to 10 seconds of incubation. This increase was mediated via an inositol 1,4,5-trisphosphate (IP(3))-dependent release of Ca(2+) from the sarcoplasmic reticulum. However, atherosclerosis is a gradual process in which VSMCs are more likely exposed to low concentrations of oxLDL over extended periods rather than acute exposures. It is very possible, therefore, that lower [oxLDL] and longer exposure times may induce a very different response with regard to regulation of [Ca(2+)](i). VSMCs were incubated with 4- to 100-fold lower [oxLDL] for up to 6 days. The conditions were not cytotoxic. Basal [Ca(2+)](i) was not altered. Surprisingly, however, after chronic exposure to oxLDL, a brief addition of oxLDL (0.1 mg/mL) or norepinephrine failed to elicit the expected rise in Ca(2+)(i). Because the acute effects of oxLDL on control cells were mediated through an IP(3)-dependent pathway, we investigated the integrity of the VSMC IP(3) receptors. Immunocytochemical analysis and Western blots revealed a depression in the density of IP(3) receptors after chronic exposure of VSMCs to oxLDL. These changes in IP(3) receptors have significance under atherosclerotic conditions as well. Immunocytochemical analysis revealed a decrease in IP(3) receptor density in the medial layer under atherosclerotic plaques in situ. Our data, therefore, demonstrate a striking difference between the acute and chronic effects of oxLDL on VSMC calcium. Whereas acute exposure to oxLDL stimulates [Ca(2+)](i), chronic exposure results in depressed Ca(2+) transients, apparently through a decrease in IP(3) receptor density. These changes have functional implications for the atherosclerotic vessel in vivo, and our data implicates oxLDL in this process.

The importance of lipid solubility in antioxidants and free radical generating systems for determining lipoprotein proxidation.

The oxidative modification of low-density lipoprotein (LDL) plays an important role in atherosclerosis. Protecting LDL from oxidation has been shown to reduce the risk of coronary heart disease. In this study, we compared the protective effects of two lipophilic antioxidants (vitamin E and lazaroid) with two hydrophilic antioxidants (trolox and vitamin C) in the presence of several different free radical generating systems. Vitamin E (IC50 = 5.9 microM) and lazaroid (IC50 = 5.0 microM) were more effective in inhibiting lipid peroxidation caused by a Fe-ADP free radical generating system than vitamin C (IC50 = 5.2 x 10(3) microM) and trolox (IC5 = 1.2 x 10(3) microM). Preincubation of lipoproteins with a lipophilic antioxidant increased the protective effect against various free radicals. Preincubation with hydrophilic antioxidants did not have an effect. We also tested the efficacy of the antioxidants when the free radicals were generated within the lipid or the aqueous environment surrounding the LDL. For this purpose, we used the peroxyl generating azo-compounds AMVN (2,2'-azobis(2,4-dimethylvaleronitrile)) and AAPH (2,2'azobis(2-amidinopropane) dihydrochloride). All of the antioxidants tested were more effective against free radicals generated in a water soluble medium than they were against free radicals generated in a lipid environment. In conclusion, our data demonstrate that lipid solubility is an important factor for both the antioxidant and the free radical generating systems in determining the extent of lipid peroxidation in LDL. Our data also demonstrate that antioxidant efficacy in one set of experimental conditions may not necessarily translate into a similar degree of protection in another set of conditions where lipophilicity is a variable.

Susceptibility of LDL to oxidative stress in obstructive sleep apnea.

Cardiovascular diseases are more common in patients with obstructive sleep apnea (OSA) than in the general population. We hypothesized that severe hypoxemia during sleep in these patients may cause an imbalance between reactive oxygen species and the antioxidant reserve that is important for the detoxification of these molecules. We tested the hypothesis that low-density lipoproteins (LDL) in hypoxic OSA patients may be more susceptible to oxidative stress than LDL of nonhypoxic OSA patients and normal controls. Fifteen OSA patients were included in this study, six with severe hypoxia (hypoxic group) who spent more than 10 minutes during sleep with SaO2 < 85% (mean 96 minutes), and nine OSA patients (nonhypoxic group) who spent less than 10 minutes during sleep with SaO2 < 85% (mean 1.1 minutes). Six healthy nonsmoking males of the same age group were included as a control group. The susceptibility of each individual's LDL to oxidative stress was examined after free-radical challenge in vitro by assessing changes in levels of conjugated dienes. The LDL in OSA patients with severe hypoxia was not more susceptible to oxidative stress compared to the LDL of nonhypoxic OSA patients and normal controls. After 6 hours of exposure to an oxidative agent, the changes in the mean conjugated diene were not different among the three groups (p = 0.75). The time required to reach 50% of maximal absorbance was also not different, p = 0.199. Glutathione peroxidase and catalase activities in red blood cells in the hypoxic and nonhypoxic patient groups were not significantly different. One night of CPAP therapy in each patient group did not significantly change the level of the antioxidant enzymes. Our results did not show any difference in the susceptibility to oxidative stress between hypoxic and nonhypoxic OSA patients and normal controls.

Oxidation of nuclear membrane cholesterol inhibits nucleoside triphosphatase activity.

Oxygen derived free radicals can oxidize membrane cholesterol. We have previously shown that cholesterol in the nuclear membrane can modulate nuclear nucleoside triphosphatase (NTPase) activity. Nucleocytoplasmic transport of peptides and mRNA via the nuclear pore complex may be regulated by the NTPase. The purpose of the present study was to determine if oxidation of nuclear cholesterol could alter NTPase activity. Nuclear membrane cholesterol was oxidized in situ with cholesterol oxidase (to selectively oxidize cholesterol) and NTPase activity measured. HPLC analysis confirmed the formation of cholesterol oxides. The activity of the NTPase was strikingly inhibited by cholesterol oxidase treatment. The Vmax of the NTPase was significantly decreased after cholesterol oxidase treatment but the Km value was unchanged. The sensitivity of NTPase activity to varying cholesterol oxidase concentrations also suggested that cholesterol located in the inner leaflet of the nuclear membrane appeared to be more important in the modulation of NTPase activity than that in the cytoplasmic leaflet. Our results indicate that oxidation of nuclear membrane cholesterol inhibits NTPase activity. These results have implications for peptide and mRNA flux across the nuclear membrane during conditions where lipid oxidation may be expected.

The presence and partitioning of calcium binding proteins in hepatic and cardiac nuclei.

Calcium is an important signal in key nuclear events, including cell cycle timing regulation of gene expression and activation of nuclear kinases and phosphatases. It is therefore important to identify calcium binding proteins in the nucleus which may play roles in these functions, and to determine whether these proteins are located in the nuclear envelope or in the nucleoplasm. Rat hepatic and pig cardiac nuclei were isolated and treated with a high salt solution to separate nucleoplasmic proteins from those associated with the nuclear envelope. The presence of calcium binding proteins was then revealed by Stains-All, staining of electrophoretic gels and 45Ca2+ overlays of Western blots. Four major calcium binding proteins were putatively identified in the pig cardiac nuclei, and another three in the rat hepatic nuclei. Proteins of 110, 93 and 35 kDa were observed in the pig cardiac nuclear envelope fraction, and another of 55 kDa in the pig cardiac high salt fraction. A 93-kDa protein was observed in the rat hepatic nuclear envelope fraction, and others of 120 and 110 kDa in the rat hepatic high salt fraction. A tentative identification has been made of the 93-kDa protein in each tissue type as calnexin, and of the cardiac 55 kDa protein as calsequestrin. This study, therefore, has putatively identified for the first time the presence of several calcium binding proteins which have distinct partitioning within hepatic and cardiac nuclei. This localization may play an important functional role within the nuclei.

Adult cardiomyocytes express functional high-affinity receptors for basic fibroblast growth factor.

As a first step in addressing the question of function for basic fibroblast growth factor (bFGF) in the adult myocardium, expression of bFGF receptors by adult rat myocytes was investigated. Cross-linking of 125I-labeled bFGF to purified sarcolemmal vesicles from adult hearts indicated specific binding to 90- to 104-kDa proteins, whereas equilibrium binding studies revealed the presence of "low"-affinity (1 nM) and "high"-affinity (115 pM) sites. Adult myocytes were found to express short and long variants of bFGF receptor 1 (FGFR-1, tyrosine kinase) mRNA. Adult heart overall levels of FGFR-1 mRNA were decreased by about one-third of corresponding fetal values. Several lines of evidence indicated that bFGF receptors in adult cardiomyocytes in situ and/or in isolation are functional. Isolated adult myocytes were found to be capable of heparin-resistant internalization of 125I-labeled bFGF, to lose their viability after interaction with bFGF-saporin (a mitotoxin known to kill cells after entry via the bFGF receptor), and to respond to bFGF by activation of mitogen-activated protein kinase. In addition, introduction of exogenous bFGF into the myocardium by Langendorff perfusion resulted in stimulation of tyrosine phosphorylation in association with cardiomyocyte intercalated disks, as assessed by immunofluorescence. It is concluded that adult cardiomyocytes express functionally coupled high-affinity bFGF receptors and that they are capable of a biologic response to bFGF in vivo.

Involvement of lipoproteins, free radicals, and calcium in cardiovascular disease processes.

Low density lipoproteins have the capacity to alter cell calcium concentrations of a wide variety of cell types. The precise mechanism of physical interaction of the LDL or its products with the cell is not defined yet. There is more agreement about the calcium transport pathway affected. Most studies show an ability of LDL to alter transsarcolemmal calcium flux, probably through the slow calcium channel. However, internal stores of calcium like the sarcoplasmic reticulum also appear to be altered. These effects of LDL on cellular calcium homeostasis have potential importance in both physiological and disease settings. Oxidised LDL has even more potential significance than the native LDL in disease conditions. Free radical mediated oxidation of LDL during atherosclerotic and ischaemic conditions creates a molecule with even greater potency for altering cell calcium and ultimately cell function and viability. The interaction of this trio of compounds--LDL, free radicals, and cellular calcium--may be critical for the mechanism of pathogenesis in specific cardiovascular diseases. It will be important in the future to identify new therapeutic strategies to prevent their interaction.

Modulation of cardiac performance by amiloride and several selected derivatives of amiloride.

Amiloride and its derivatives (benzamil, dichlorobenzamil, 5-(N,N-dimethyl)-amiloride, 5-(N-ethyl-N-isopropyl)-amiloride, (N,N-hexamethylene)- amiloride and 5-(N-methyl-N-isobutyl)-amiloride) are commonly used as selective blockers of Na+/Ca++ exchange or Na+/H+ exchange. Very little information is currently available regarding their effects on cardiac performance. It was observed that addition of amiloride or any of the selected derivatives to the coronary perfusate of the right ventricular wall produced a potent depressive effect on peak developed tension and the rates of tension generation and dissipation. The concentrations at which this occurred are those that are commonly used in ischemia or hypoxia studies. Significantly, the depressive action of the drugs increased with the perfusion duration and never achieved a stable level. An initial, transient positive inotropic effect was observed with some of the drugs. If the drug concentration and perfusion time was limited, the effects were reversible. All of the drugs except amiloride produced extra systoles. The drugs were capable of blocking Ca++ transients in isolated cardiomyocytes but had little effect on intracellular pH. The drugs lengthened the action potential duration and decreased the action potential amplitude and upstroke velocity. Their effects on cardiac performance may involve a complex inhibition of Ca++ influx and K+ efflux in addition to a stimulation of a nonselective cation current. It is concluded that amiloride and its analogs have striking effects on cardiac performance which may be unrelated to their capacity to inhibit Na+/Ca++ or Na+/H+ exchange. In summary, the use of these drugs is not normally recommended in cell or tissue perfusion experiments because of their nonselectivity. However, if the drug concentration and perfusion time is controlled carefully, interpretable data may be obtained in some cases.

The action of oxidized low density lipoprotein on calcium transients in isolated rabbit cardiomyocytes.

The effect of oxidized low density lipoprotein (oxLDL) on the Ca2+ transients of isolated rabbit cardiomyocytes was assessed. LDL was oxidized by a free radical generating system in vitro. The systolic Ca2+ concentration in transients was significantly increased after treatment with 100 micrograms of oxLDL cholesterol/ml for 16 min without having any effect on the diastolic [Ca2+]. However, a toxic effect was observed when the concentration of oxLDL was increased to 1,000 micrograms/ml. Cells treated with 100 micrograms of oxLDL/ml were more sensitive to the blocking action of nicardipine on the Ca2+ transient than were control cardiomyocytes. Furthermore, oxLDL failed to produce a stimulatory effect on the transient when the extracellular [Ca2+] was lowered. The malondialdehyde content in the oxLDL correlated well with the change in systolic [Ca2+] of treated cells. Lazaroid, a novel antioxidant, completely inhibited malondialdehyde formation in the oxLDL and prevented the increment of systolic [Ca2+] in the treated cells. The data indicate that oxLDL can induce relatively rapid alterations in cellular Ca2+ transients via a modification of Ca2+ entry through the L-type Ca2+ channel. LDL oxidation induced by free radicals may play an important role in influencing cardiac contractile function during pathological conditions such as ischemia/reperfusion challenge.