Impaired myocardial perfusion reserve in experimental hypercholesterolemia is independent of myocardial neovascularization

Our objective was to investigate the functional role of hypercholesterolemia-associated myocardial neovascularization in early atherosclerosis using the antiangiogenic thalidomide. Experimental atherosclerosis is characterized by myocardial neovascularization, associated with a decrease in myocardial perfusion response to challenge, coronary endothelial dysfunction, and high oxidative stress. However, the functional significance of these neovessels is not known. Three groups of pigs (n = 6 each) were studied after 12 wk of normal or hypercholesterolemic diet without (HC) or with thalidomide (HC + Thal). Myocardial perfusion and permeability were assessed at baseline and in response to cardiac challenge, using electron beam computed tomography, and coronary endothelial function was assessed using organ chambers. Myocardial samples were scanned ex vivo with a three-dimensional microscopic computed tomography scanner, and the spatial density of the myocardial microvessels was quantified. Growth factors and oxidative stress were measured in the myocardial tissue. As a results of these procedures, myocardial perfusion response to adenosine and dobutamine was blunted in both HC and HC + Thal pigs compared with normal pigs (P < 0.05, HC and HC + Thal vs. normal) as was the coronary endothelial function. Myocardial permeability response to adenosine was increased in both HC and HC + Thal pigs compared with normal pigs (P < 0.05, HC and HC + Thal vs. normal, and HC + Thal vs. HC). The microvascular density was increased in HC pigs compared with normal pigs but normalized in HC + Thal pigs (P < 0.001 HC vs. normal and HC + Thal). HC + Thal pigs showed decreased expression of Flk-1 and basic FGF but increased expression of VEGF compared with normal and HC pigs. Oxidative stress was increased in both HC and HC + Thal pigs compared with normal pigs. In conclusion, chronic administration of thalidomide attenuates myocardial neovascularization in experimental HC pigs without affecting myocardial perfusion response to stimulation. This suggests that the myocardial neovascularization may not contribute to the attenuated myocardial perfusion response in hypercholesterolemia.     

Receptors for pea and lentil lectins on human lymphoid cells: demonstration of distinct lectin-defined cell subclasses

Lectins are useful probes for studying cell surface glycoconjugates. Pea (PL) and lentil (LL) lectin each requires for binding a fucosyl- and two alpha-mannosyl residues in core regions of glycopeptides, but differences in outer chain carbohydrates may alter their relative binding affinities. We used binding studies with [125I]-PL and LL and flow cytometry with fluorescein-conjugated (FITC)-PL and -LL to study their interactions with peripheral lymphocytes. Binding of both lectins to lymphocytes was saturable, reversible, and inhibited by alpha-methyl mannose. Scatchard analyses were consistent with two classes of receptors for each lectin. Flow cytometric analyses demonstrated that cell to cell receptor densities varied. Sixty-five percent of lymphocytes bound PL (mean 2 X 10(6) receptors/cell) and 45% bound LL (mean 3 X 10(6) receptors/cell). Competition studies demonstrated mutual inhibition, but flow cytometry revealed persistent FITC-PL or -LL binding depsite 20-fold molar excess of the other lectin. Distributions of receptors for PL and LL on lymphocytes were as follows: 45% of lymphocytes bound both PL and LL; 20% of lymphocytes bound PL alone; 35% of lymphocytes bound neither PL nor LL. Despite similar binding requirements for PL and LL and overlap between their receptors on lymphocytes, there appear to be subsets of receptors specific for each lectin. These results may reflect abilities of PL and LL to discriminate subtle carbohydrate differences on lymphocyte surfaces.     

Transforming growth factor-beta (TGF-beta) type I receptor/ALK5-dependent activation of the GADD45beta gene mediates the induction of biglycan expression by TGF-beta

We have recently shown that induction of biglycan (BGN) expression by transforming growth factor-beta1 (TGF-beta1) required sequential activation of both Smad and p38 mitogen-activated protein kinase signaling (Ungefroren, H., Lenschow, W., Chen, W.-B., and Kalthoff, H. (2003) J. Biol. Chem. 278, 11041-11049). Here, we have analyzed the receptors through which TGF-beta1 controls expression of BGN and GADD45beta, the latter of which is postulated to link early Smad signaling to delayed activation of p38. Ectopic expression of a dominant-negative mutant of the TGF-beta type II receptor in PANC-1 cells abrogated TGF-beta-induced BGN up-regulation. Similarly, inhibition of the TGF-beta type I receptor/ALK5 with either SB431542 or by enforced stable expression of a kinase-dead mutant greatly attenuated the TGF-beta effect on both BGN and GADD45beta expression in PANC-1 and MG-63 cells. The enhancing effect of ALK5 on TGF-beta-mediated GADD45beta and BGN expression and on GADD45beta promoter activity was also dependent on its ability to activate Smad signaling, because an ALK5 mutant defective in Smad activation (TbetaRImL45) but with an otherwise functional kinase domain failed to mediate these responses. The TGF-beta/ALK5 effect on p38 activation and BGN expression was mimicked by overexpression of GADD45beta alone (in the absence of TGF-beta stimulation) and suppressed upon antisense inhibition of GADD45beta expression. These results show that TGF-beta induces BGN expression through (the Smad-activating function of) ALK5 and GADD45beta and suggest that the sensitivity of MyD118 to activation by TGF-beta, which varies between tissues, ultimately determines the strength of the TGF-beta effect on BGN.     

Opposite effect of NF-kappa B and c-Jun N-terminal kinase on p53-independent GADD45 induction by arsenite

Cell cycle checkpoint, a major genomic surveillance mechanism, is an important step in maintaining genomic stability and integrity in response to environmental stresses. Using cells derived from human bronchial epithelial cells, we demonstrate that NF-kappaB and c-Jun N-terminal kinase (JNK) reciprocally regulate arsenic trioxide (arsenite)-induced, p53-independent expression of GADD45 protein, a cell cycle checkpoint protein that arrests cells at the G(2)/M phase transition. Inhibition of NF-kappaB activation by stable expression of a kinase-mutated form of IkappaB kinase caused increased and prolonged induction of GADD45 by arsenite. In contrast, the induction of GADD45 by arsenite was transient and less potent in cells where the NF-kappaB activation pathway was normal. Analysis of the cell cycle profile by flow cytometry indicated that NF-kappaB inhibition potentiates arsenite-induced G(2)/M cell cycle arrest. Abrogation of JNK activation, on the other hand, decreased GADD45 expression induced by arsenite, suggesting a role for JNK activation in GADD45 induction. These results indicate a molecular mechanism by which NF-kappaB and JNK may differentially contribute to cell cycle regulation in response to arsenite.     

p53-independent induction of GADD45 and GADD153 in mouse lungs exposed to hyperoxia

Previous studies have shown that lungs of adult mice exposed to >95% oxygen have increased terminal deoxyribonucleotidyltransferase dUTP nick end-label staining and accumulate p53, the expression of which increases in cells exposed to DNA-damaging agents. The present study was designed to determine whether hyperoxia also increased expression of the growth arrest and DNA damage (GADD) gene 45 and GADD153, which are induced by genotoxic stress through p53-dependent and -independent pathways. GADD proteins have been shown to inhibit proliferation and stimulate DNA repair and/or apoptosis. GADD45 and GADD153 mRNAs were not detected in lungs exposed to room air but were detected after 48 and 72 h of exposure to hyperoxia. In situ hybridization and immunohistochemistry revealed that hyperoxia increased GADD45 and GADD153 expression in the bronchiolar epithelium and GADD45 expression predominantly in alveolar cells that were morphologically consistent with type II cells. Hyperoxia also increased GADD expression in p53-deficient mice. Terminal deoxyribonucleotidyltransferase dUTP nick end-label staining of lung cells from p53 wild-type and p53-null mice exposed to hyperoxia for 48 h revealed that hyperoxia-induced DNA fragmentation was not modified by p53 deficiency. These studies are consistent with the hypothesis that hyperoxia-induced DNA fragmentation is associated with the expression of GADD genes that may participate in DNA repair and/or apoptosis.     

Effects of exercise training and hypercholesterolemia on adenosine activation of voltage-dependent K+ channels in coronary arterioles

Coronary arterioles from hypercholesterolemic swine display attenuated adenosine-mediated vasodilatation that is attributable to the elimination of voltage-dependent K(+) (Kv) channel stimulation. For the present study, we tested the hypotheses that exercise training would correct impaired adenosine-induced dilatation in coronary arterioles from hypercholesterolemic pigs through restoration of adenosine activation of Kv channels and that vasodilatation to the receptor-independent adenylyl cyclase activator, forskolin, would also be attenuated in arterioles from hypercholesterolemic pigs. Pigs were randomly assigned to a control (NC) or high-fat, high-cholesterol (HC) diet for 20 wk. Four weeks after the diet was initiated, pigs from both groups were assigned to exercise training (Ex; 5 days/wk for 16 wk) or sedentary (Sed) protocols, resulting in four groups of pigs: NC-Sed, NC-Ex, HC-Sed, and HC-Ex. Arterioles ( approximately 150 mum) from both HC-Sed and HC-Ex pigs displayed impaired adenosine-mediated dilatation that was attributable to the elimination of 4-aminopyridine (4-AP; 1 mM)-sensitive Kv channel activation compared with NC counterparts. Arteriolar smooth muscle whole cell Kv currents were significantly reduced in HC-Sed compared with NC-Sed, although HC-Ex and NC-Ex did not differ. Forskolin-mediated dilatation was attenuated by 4-AP (1 mM) and in a concentration-dependent manner by tetraethylammonium (TEA; 0.1-1 mM) in NC-Sed but not HC-Sed. Further, TEA-sensitive Kv currents were diminished in cells of HC-Sed compared with NC-Sed pigs. Quantitative RT-PCR revealed similar expression levels of Kv3.1 and 3.3 in arterioles of NC-Sed and HC-Sed swine with undetectable expression of Kv1.1, 3.2, and 3.4. Taken together, these results suggest that hypercholesterolemia-mediated attenuation of adenosine-induced vasodilatation in coronary arterioles is not corrected by exercise training and is likely attributable to an impairment in the pathway coupling adenylyl cyclase with a highly TEA-sensitive Kv channel isoform(s).     

Early changes in coronary artery wall structure detected by microcomputed tomography in experimental hypercholesterolemia

Changes in the structure of the artery wall commence shortly after exposure to cardiovascular risk factors, such as hypercholesterolemia (HC), but may be difficult to detect. The ability to study vascular wall structure could be helpful in evaluation of the factors that instigate atherosclerosis and its pathomechanisms. The present study tested the hypothesis that early morphological changes in coronary arteries of hypercholesterolemic (HC) pigs can be detected using the novel X-ray contrast agent OsO(4) and three-dimensional micro-computed tomography (CT). Two groups of pigs were studied after they were fed a normal or an HC (2% cholesterol) diet for 12 wk. Hearts were harvested, coronary arteries were injected with 1% OsO(4) solution, and cardiac samples (6-mum-thick) were scanned by micro-CT. Layers of the epicardial coronary artery wall, early lesions, and perivascular OsO(4) accumulation were determined. Leakage of OsO(4) from myocardial microvessels was used to assess vascular permeability, which was correlated with immunoreactivity of vascular endothelial growth factor in corresponding histological cross sections. OsO(4) enhanced the visualization of coronary artery wall layers and facilitated detection of early lesions in HC in longitudinal tomographic sections of vascular segments. Increased density of perivascular OsO(4) in HC was correlated with increased vascular endothelial growth factor expression and suggested increased microvascular permeability. The use of OsO(4) as a contrast agent in micro-CT allows three-dimensional visualization of coronary artery wall structure, early lesion formation, and changes in vascular permeability. Therefore, this technique can be a useful tool in atherosclerosis research.     

The c-Myc Oncoprotein Interacts with Bcr

Bcr is a multifunctional protein that is the fusion partner for Abl (p210 Bcr-Abl) in Philadelphia chromosome positive leukemias. We have identified c-Myc as a binding partner for Bcr in both yeast and mammalian cells. We are also able to observe interactions between natively expressed c-Myc and Bcr in leukemic cell lines. Although Bcr and Max have overlapping binding sites on c-Myc, Bcr cannot interact with Max, or with the c-Myc.Max heterodimer. Bcr expression blocks activation of c-Myc-responsive genes, as well as the transformed phenotype induced by coexpression of c-Myc and H-Ras, and this finding suggests that one function of Bcr is to limit the activity of c-Myc. However, Bcr does not block c-Myc function by preventing its nuclear localization. Interestingly, increased Bcr dosage in COS-7 and K-562 cells correlates with a reduction in c-Myc protein levels, suggesting that Bcr may in fact be limiting c-Myc activity by regulating its stability. These data indicate that Bcr is a novel regulator of c-Myc function whose disrupted expression may contribute to the high level of c-Myc protein that is observed in Bcr-Abl transformed cells.     

Role of vasa vasorum in transendothelial solute transport in the coronary vessel wall: a study with cryostatic micro-CT

Using cryostatic microscopic computed tomography (micro-CT), we sought to determine the role of coronary vasa vasorum (VV) in transendothelial solute transport in arteries with normal and increased permeability due to high plasma cholesterol levels. In 6-mo-old pigs on a normal (n=23) and 2% high cholesterol (HC) diet (n=8), 2-cm segments of the proximal left anterior descending coronary arteries were removed in vivo after a selective injection of X-ray contrast solution. Harvesting of the specimens occurred at 0, 15, 25, 35, or 45 s after completion of the contrast injection. Specimens were snap frozen and scanned in our cryostatic micro-CT. The spatial distribution of contrast in the coronary artery wall was quantified using the CT images. Right coronary arteries were infused with Microfil to determine VV density (VV/mm2) and the cumulative lumen surface area (mm2/mm3). Transendothelial diffusion of contrast into the coronary vessel wall is a dynamic process starting at both the subintima and the adventitia. The subintimal opacification moves as a wave toward the adventitia, whereas the adventitial wave resolves. The coronary vessel wall in animals on a HC diet shows higher opacification than in normal coronary arteries without an increase of VV total luminal surface area. The loss of endothelial integrity in hypercholesterolemia significantly alters VV solute washin to, and washout from, the coronary artery wall.