Regulation of vascular smooth muscle cell apoptosis. Modulation of bad by a phosphatidylinositol 3-kinase-dependent pathway.

Our objective was to define the signaling mechanisms by which mitogens such as insulin-like growth factor-I (IGF-I) regulate vascular smooth muscle cell (VSMC) apoptosis. We confirmed that IGF-I inhibits serum withdrawal-induced apoptosis of cultured VSMCs in a dose-dependent and time-dependent fashion. To test the hypothesis that the phosphatidylinositol (PI) 3-kinase signaling pathway regulates VSMC survival, we examined the relationship between PI 3-kinase activity and cell fate. PI 3-kinase was elevated in viable VSMCs maintained in serum-containing medium, declined significantly in response to serum withdrawal, and increased in response to IGF-I-induced survival. Moreover, blockade of PI 3-kinase with 2 structurally dissimilar inhibitors (wortmannin or LY294002) abolished the capacity of IGF-I to maintain VSMC viability. Similarly, transient transfection of a dominant-negative Deltap85 PI 3-kinase mutant construct abrogated the capacity of IGF-I to prevent VSMC death. Thus, PI 3-kinase is a critical antiapoptotic signal in VSMCs. To define the distal element of the antiapoptotic cascade, we tested the hypothesis that IGF-I inhibits the influence of the proapoptotic gene Bad. Indeed, IGF-I stimulates increased expression of the inactive, phosphorylated form of Bad by a PI 3-kinase-dependent pathway. Moreover, the proapoptotic effect of Bad was attenuated by the stimulation of IGF-I. Thus, growth factors appear to prevent VSMC death by activating signal transduction pathways linked to apoptotic regulatory genes.

Vascular cell apoptosis: cell type-specific modulation by transforming growth factor-beta1 in endothelial cells versus smooth muscle cells.

BACKGROUND: It is postulated that vascular lesion formation and remodeling involves a balance between vascular cell death and cell proliferation. Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic factor expressed within vascular cells that regulates cell growth in a tissue-specific manner. This study tested the hypothesis that the control of vascular cell apoptosis involves cell type-specific regulation by TGF-beta1. METHODS AND RESULTS: In response to serum withdrawal, cultured endothelial cells and vascular smooth muscle cells exhibited apoptosis as evidenced by DNA laddering and quantitated by analysis of nuclear chromatin morphology. Addition of TGF-beta1 to endothelial cells in serum-free media further potentiated the induction of apoptosis in a dose-dependent fashion. Moreover, TGF-beta1 promoted endothelial cell death despite the presence of 10% serum. However, endothelial cells plated on collagen I were resistant to TGF-beta1-induced apoptosis. This antiapoptotic influence of the matrix was mimicked by integrin activation with anti-beta1 antibodies and associated with increased expression of the antiapoptotic factor bcl-2. In accord with the hypothesis that the modulation of antiapoptotic gene expression may mediate the effects of TGF-beta1 and beta1 integrins on cell fate, we observed that endothelial cells with constitutive upregulation of bcl-2 remained viable despite exposure to TGF-beta1 in serum-free conditions. In contrast to the proapoptotic effect of TGF-beta1 in endothelial cells, addition of TGF-beta1 to vascular smooth muscle cells in serum-free media inhibited apoptosis. CONCLUSIONS: These findings suggest that the effect of cytokines such as TGF-beta1 on cell fate is contextual and is modulated by cell-matrix interactions in a cell type-specific manner.

Regression of atherosclerosis: role of nitric oxide and apoptosis.

BACKGROUND: We have recently found that administration of L-arginine to hypercholesterolemic rabbits induces regression of preexisting lesions. Others have previously shown that activation of the L-arginine/nitric oxide (NO) synthase pathway can induce apoptosis of vascular cells in vitro. Accordingly, the current study was designed to determine if dietary supplementation of L-arginine induces apoptosis of intimal lesions and if this effect is mediated through the NO synthase pathway. METHODS AND RESULTS: Male New Zealand White rabbits were fed a 0.5% cholesterol diet for 10 weeks and subsequently placed on 2.5% L-arginine HCl in the drinking water, and the cholesterol diet was continued for 2 weeks, at which time the aortas were harvested for histological studies. L-Arginine treatment increased the number of apoptotic cells (largely macrophages) in the intimal lesions by 3-fold (11.9+/-3.9 vs 3.9+/-1. 4 apoptotic cells/mm2, P<0.01). In subsequent studies, aortas were harvested for ex vivo studies. Aortic segments were incubated in cell culture medium for 4 to 24 hours with modulators of the NO synthase pathway. The tissues were then collected for histological studies and the conditioned medium collected for measurement of nitrogen oxides by chemiluminescence. Addition of sodium nitroprusside (10(-5) mol/L) to the medium caused a time-dependent increase in apoptosis of vascular cells (largely macrophages) in the intimal lesion. L-Arginine (10(-3) mol/L) had an identical effect on apoptosis, which was associated with an increase in nitrogen oxides released into the medium. These effects were not mimicked by D-arginine, and they were antagonized by the NO synthase inhibitor L-nitro-arginine (10(-4) mol/L). The effect of L-arginine was not influenced by an antagonist of cGMP-dependent protein kinase, nor was the effect mimicked by the agonist of protein kinase G or 8-BR cGMP. CONCLUSIONS: These results indicate that supplemental L-arginine induces apoptosis of macrophages in intimal lesions by its metabolism to NO, which acts through a cGMP-independent pathway. These studies are consistent with our previous observation that supplementation of dietary arginine induces regression of atheroma in this animal model. These studies provide a rationale for further investigation of the therapeutic potential of manipulating the NO synthase pathway in atherosclerosis.

Endothelial cell apoptosis in capillary network remodeling.

We hypothesized that the regulation of apoptosis is an important determinant of capillary network structure. Using human umbilical vein endothelial cells (HUVEC) in in vitro model systems of capillary tube formation, we initially documented that apoptosis is a prominent feature of network formation. Perturbations of integrin-matrix signaling by the administration of either colchicine or an anti-alpha(v)beta3 antibody resulted in the dissolution of the tubular network in association with increased apoptosis. The activation of the alpha(v)beta3 integrin induced increased expression of the anti-apoptotic gene bcl-2 and conferred resistance to the proapoptotic effect of TGF-beta1. In contrast to the stable networks formed by HUVEC, bovine aortic endothelial cells (BAEC) exhibited a more dynamic process of network formation and spontaneous involution. The inhibition of BAEC apoptosis by stable transfection of bcl-2 prevented the involution of the network. We hypothesized that TGF-beta1 present within the model system mediated network involution by inducing BAEC death. Indeed, blockade of TGF-beta1 with neutralizing antibodies reduced BAEC apoptosis and preserved the network structure. As observed with HUVEC networks, stable BAEC networks formed during blockade of TGF-beta1 were also dependent on the survival-promoting effects of matrixintegrin interactions. This study suggests that capillary network structure is determined by the balance of proapoptotic vs. anti-apoptotic signals mediated by the engagement of cytokine and integrin receptors within the milieu.

Determinants of vascular smooth muscle cell apoptosis after balloon angioplasty injury. Influence of redox state and cell phenotype.

We have observed that acute medial cell loss is an initial event in the response to vascular injury induced by balloon-catheter distention of the rabbit carotid artery. Numerous apoptotic medial cells were observed as early as 30 minutes after balloon inflation, and a 70% loss of cellularity was evident by 90 minutes. Balloon injury was associated with oxidative stress as reflected by a fall in glutathione levels by 63% within 30 minutes after injury. We hypothesized that balloon injury activated a redox-sensitive signaling pathway coupled to the regulation of apoptosis. Indeed, the activity of the proapoptotic signal mediator, stress-activated protein kinase, was increased severalfold within 10 minutes after injury. Moreover, modifying the vascular redox state by the administration of 1 of 2 structurally dissimilar antioxidants, N-acetyl cysteine or pyrrolidine dithiocarbamate, markedly attenuated both stress-activated protein kinase activation and the induction of apoptosis at 30 minutes. We hypothesized further that the induction of vascular smooth muscle cell apoptosis is modulated by phenotype. In contrast to medial cells, we observed that neointimal cells were relatively resistant to apoptotic death induced by angioplasty injury. This resistance to balloon injury-induced death was associated with an upregulation of the antiapoptotic mediator bcl-xL. This study suggests that acute apoptotic cell death after vascular injury is a highly regulated process governed by cellular redox state and the relative expression of antiapoptotic genes. Angioplasty-induced vascular cell apoptosis may be an important determinant of vascular remodeling and restenosis.

Angiotensin II type 2 receptor mediates vascular smooth muscle cell apoptosis and antagonizes angiotensin II type 1 receptor action: an in vitro gene transfer study.

Angiotensin II type 2 (AT2) receptor is expressed abundantly in the fetal vasculature with rapid decline after birth and re-expressed in the adult vasculature after injury, whereas angiotensin II type 1 (AT1) receptor is expressed. We studied their effects on apoptosis in cultured rat vascular smooth muscle cells (VSMC). Serum starvation induced VSMC DNA fragmentation and the stimulation of AT1 receptor inhibited this apoptotic change. We transfected rat AT2 receptor cDNA, since cultured adult VSMCs show very low level of endogenous AT2 receptor. In AT2 receptor transfected VSMC, selective stimulation of AT2 receptor facilitated serum-deprivation-induced apoptosis and AT1 receptor stimulation inhibited it. Moreover we observed that AT1 receptor stimulation activated extracellular signal-regulated kinase (ERK), whereas the AT2 receptor stimulation inhibited the activation of ERK. Taken together, our results suggest that AT1 and AT2 receptors exert counteracting effects on ERK activation and consequently VSMC apoptosis and differential expression of these receptors may participate in vascular development and vascular remodeling.

Use of assumed versus measured oxygen consumption for the determination of cardiac output using the Fick principle.

Assumed oxygen consumption (VO2) is increasingly used as a convenient surrogate for measured VO2 for calculation of cardiac output. This substitution is often based on empirical formulae, previously validated only in relatively young patients. To assess the inaccuracy introduced by extrapolating these formulae to older patients, we compared measured VO2 with assumed VO2 in 57 patients. VO2 was measured using an open circuit analyzer. Assumed VO2 was calculated according to the LaFarge or Bergstra formulae. Agreement between both methods was assessed according to the method of Bland and Altman. The mean difference of measured VO2 minus assumed VO2 was 7.9 ml/min/m2 (P < 0.02) using the LaFarge formula, and -15.6 ml/min/m2 (P < 0.0002) using the Bergstra formula across a range of measured VO2 from 70 to 176 ml/min/m2. A systematic error was introduced by assumed VO2 from both formulae of underestimating higher and overestimating lower values of VO2, resulting in poor overall agreement with measured VO2. The same error and poor agreement was found when analyzing subgroups of patients > or =60 or <70 years of age. In summary, use of assumed VO2 introduces large, unpredictable errors in adult patients, suggesting requirement for measurement of VO2 when calculating cardiac output.

Inhibition of neointimal cell bcl-x expression induces apoptosis and regression of vascular disease.

We postulated that activation of a genetic program that tonically inhibits intimal cell death is a necessary condition for the pathogenesis of vascular disease. Studies of vascular lesions in humans and animal models documented increased expression of the anti-apoptotic gene product Bcl-xL within intimal cells. Downregulation of intimal cell bcl-xL expression with the use of antisense oligonucleotides induced apoptosis and acute regression of vascular lesions. These findings indicate that apoptosis regulatory genes such as bcl-xL are critical determinants of intimal lesion formation and that targeted apoptosis may be a novel therapy for intimal vascular disease.

Vasoactive substances regulate vascular smooth muscle cell apoptosis. Countervailing influences of nitric oxide and angiotensin II.

This study tests the hypothesis that the control of vascular smooth muscle cell (VSMC) apoptosis is regulated by the antagonistic balance between vasoactive substances such as NO and angiotensin II (Ang II). Moreover, it is postulated that the cellular signaling pathways involved in regulating vessel tone are also coupled to the regulation of programmed cell death. Using an in vitro model system, we documented that the addition of NO donor molecules S-nitroso-N-acetylpenicillamine or sodium nitroprusside to VSMC dose-dependently induced apoptosis as documented by DNA laddering and quantified by analysis of cellular chromatin morphology. The mediator role of the guanylate cyclase signaling pathway in NO-induced apoptosis was evidenced by (1) induction of apoptosis by the 8-bromo-cGMP analogue, (2) potentiation of NO-induced apoptosis by cGMP-specific phosphodiesterase inhibition, and (3) the prevention of NO-induced apoptosis by the inhibition of the cGMP-dependent protein kinase 1 alpha. In contrast, Ang II directly antagonized NO donor- and cGMP analogue-induced apoptosis via activation of the type I Ang II receptor. These findings suggest that the countervailing balance between NO and Ang II may determine the overall cell population within the vessel wall by regulating genetic programs determining cell death as well as cell growth.

Quantitation of elastin in tissues and culture: problems related to the accurate measurement of small amounts of elastin with special emphasis on the rat.

Both rat and sheep elastin can be quantified by measurement of discrete peptides released from the insoluble protein by thermolysin digestion. These peptides are easily visualized and measured by HPLC. With the sheep the tallest peak on the chromatogram represents the VGVPG pentapeptide derived from a repeating sequence seen in elastin from many species. This repeating sequence allows for amplification of the signal significantly above background so that accurate quantitation can be carried out. The measurement is reproducible over a wide range of protein concentrations. With the rat however the pentapeptide is not present but appears to be replaced by other repeating sequences. We quantitated and determined amino acid sequence on 8 peaks present in the early portion of the chromatogram for purposes of quantifying rat elastin. That signal most reliably present over a range of concentrations was tyrosyl-glycine (YG) which eluted at 8.5 minutes. We have used YG as a basis for quantitation of rat elastin both from tissues and tissue culture. We have also shown that the desmosine crosslinks are not constant in elastin produced in a neonatal rat smooth muscle culture system but vary with the age of the culture. We thus propose that an index of maturation be considered for a given elastin in the form of mumoles of crosslink per gram of elastin so as to better define its quality.

Rapid isolation of high-molecular-weight DNA from agarose gels.

We have developed a simple, reliable, and rapid method for recovering DNA from agarose gels. While many methods for DNA extraction have already been described, few provide quantitative recovery of large DNA molecules. These procedures generally require costly apparatus, extended elution times, or considerable handling of the sample after elution. Our method employs a novel electroelution chamber constructed from acrylic plastic. Gel slices containing DNA are placed in the chamber between platinum electrodes. Voltage is applied and a continuous flow of buffer sweeps the eluted DNA from the chamber into an external receptacle. Elution is complete in 7 min. Concentrated DNA is obtained by butanol extraction and alcohol precipitation in 1 h. Recoveries, quantitated by counting radiolabeled DNA or by densitometry of analytical gels, were 94 to 100% for fragments of 4 to 50 kb. The eluted DNA was undegraded and could be digested with restriction enzymes, ligated, end-labeled, or used to transform cells as efficiently as noneluted DNA. Complete elution of a 100-kb plasmid, a 194-kb concatemer of bacteriophage lambda, and of 440- and 550- chromosomes of Saccharomyces cerevisiae was also achieved using the same process. This method is suitable for routine use in a wide range of cloning applications, including the electrophoretic isolation of large DNA molecules.