bluuebunny/crossref_metadata_2025 huggingface dataset:
Modest actomyosin energy conservation increases myocardial postischemic function
Authors: Blunt Bradford C. , Chen Yi , Potter James D. , Hofmann Polly A.
Published: October 2004

We have proposed that pharmacological preconditioning, leading to PKC-ε activation, in hearts improves postischemic functional recovery through a decrease in actomyosin ATPase activity and subsequent ATP conservation. The purpose of the present study was to determine whether moderate PKC-independent decreases in actomyosin ATPase are sufficient to improve myocardial postischemic function. Rats were given propylthiouracil (PTU) for 8 days to induce a 25% increase in β-myosin heavy chain with a 28% reduction in actomyosin ATPase activity. Recovery of postischemic left ventricular developed pressure (LVDP) was significantly higher in PTU-treated rat hearts subjected to 30 min of global ischemia than in control hearts: 57.9 ± 6.2 vs. 32.6 ± 5.1% of preischemic values. In addition, PTU-treated hearts exhibited a delayed onset of rigor contracture during ischemia and a higher global ATP content after ischemia. In the second part of our study, we demonstrated a lower maximal actomyosin ATPase and a higher global ATP content after ischemia in human troponin T (TnT) transgenic mouse hearts. In mouse hearts with and without a point mutation at F110I of human TnT, recovery of postischemic LVDP was 55.4 ± 5.5 and 62.5 ± 14.5% compared with 20.0 ± 2.9% in nontransgenic mouse hearts after 35 min of global ischemia. These results are consistent with the hypothesis that moderate decreases in actomyosin ATPase activity result in net ATP conservation that is sufficient to improve postischemic contractile function.
Effect of sarcolemmal rupture on myocardial electrical impedance during oxygen deprivation
Authors: Rodriguez-Sinovas Antonio , García-Dorado David , Pina Pilar , Ruiz-Meana Marisol , Soler-Soler Jordi
Published: October 2004

Plasma membrane disruption is a characteristic feature of cell death induced by hypoxia or ischemia. Here, we investigated whether analysis of tissue electrical impedance allows detection of ongoing cell membrane rupture and necrotic cell death in hypoxic or ischemic myocardium. Twenty-eight isolated rat hearts were submitted to 5 h of ischemia ( n = 8) or hypoxia ( n = 20). Myocardial electrical impedance and lactate dehydrogenase (LDH) release were monitored. The time course of hypoxia-induced cell death was modified by altering pH (pH 7.4 or 6.4, 5 h) or by adding 3 or 10 mM glycine. Ischemia and hypoxia induced an increase in electrical impedance, followed by a plateau, and later a reduction. During hypoxia, LDH release started after a prolonged lapse of time (80.00 ± 8.37 min at pH 7.4 and 122.50 ± 11.82 min at pH 6.4). The onset of LDH release was followed by the onset of the late reduction in electrical impedance, and both were delayed by acidic pH ( P < 0.05) and by glycine ( P < 0.05). The times of onset of LDH release and of late electrical changes were significantly correlated ( r = 0.752, P < 0.001). In separate experiments, induction of sarcolemmal rupture with Triton X-100 ( n = 6) mimicked the late effects of ischemia or hypoxia on tissue impedance. The protective effects of glycine and acidosis on membrane disruption were confirmed (propidium iodide) in energy-deprived HL-1 cardiomyocytes. These results describe for the first time a late fall in electrical impedance in myocardium submitted to prolonged oxygen deprivation and demonstrate that this fall allows detection of ongoing cell necrosis.
Angiogenic protection from focal ischemia with angiotensin II type 1 receptor blockade in the rat
Authors: Forder Joan P. , Munzenmaier Diane H. , Greene Andrew S.
Published: October 2004

Angiogenesis within an ischemic region of the brain may increase tissue viability and act to limit the extent of an infarct. The ANG II pathway can both stimulate and inhibit angiogenesis depending on the tissue and the activated receptors. Previous work showed that 2-wk losartan administration (ANG II type 1 receptor blockade) initiates a significant cerebral angiogenic response. We hypothesized that administration of losartan in the drinking water of rats for 2 wk before initiation of focal ischemia would decrease the extent of the resulting infarct. Adult male Sprague-Dawley rats were given losartan (50 mg/day) in drinking water for 2 wk before initiation of cerebral focal ischemia produced by cauterization of cortical surface vessels. Controls received normal drinking water. In control animals, three main vessels feeding the whisker barrel cortex were cauterized, resulting in cessation of blood flow. The same protocol was followed for losartan-treated animals but did not result in cessation of blood flow in the whisker barrel cortex. Another group of losartan-treated animals received between 8 and 14 cauterizations of surface vessels feeding the whisker barrel cortex, and cessation of blood flow was verified. Rats were killed 72 h after surgery. Morphological examination revealed angiogenesis, maintained vascular delivery, and significantly decreased infarct size in losartan-treated animals compared with controls. These results demonstrate that pretreatment with losartan reduces infarct size after cerebral focal ischemia and support the hypothesis that cerebral angiogenesis may be one of the mechanisms responsible.
Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking
Authors: Brueggemann Lioubov I. , Martin Beverly L. , Barakat John , Byron Kenneth L. , Cribbs Leanne L.
Published: October 2004

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+channels in rat arterial VSM and characterize endogenous LVA Ca2+currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+spiking can be abolished by mibefradil at low concentrations (<1 μM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+currents but did not alter the AVP-stimulated Ca2+spiking response. Together these data suggest that T-type Ca2+channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+entry through these channels is not limiting for repetitive Ca2+spiking observed in A7r5 cells.
Cardiovascular actions of rattlesnake bradykinin ([Val1,Thr6]bradykinin) in the anesthetized South American rattlesnakeCrotalus durissus terrificus
Authors: Galli Gina L. J. , Skovgaard Nini , Abe Augusto S. , Taylor Edwin W. , Conlon J. Michael , Wang Tobias
Published: October 2004

Incubation of heat-denatured plasma from the rattlesnake Crotalus atrox with trypsin generated a bradykinin (BK) that contained two amino acid substitutions (Arg1→ Val and Ser6→ Thr) compared with mammalian BK. Bolus intra-arterial injections of synthetic rattlesnake BK (0.01–10 nmol/kg) into the anesthetized rattlesnake, Crotalus durissus terrificus, produced a pronounced and concentration-dependent increase in systemic vascular conductance (Gsys). This caused a fall in systemic arterial blood pressure (Psys) and an increase in blood flow. Heart rate and stroke volume also increased. This primary response was followed by a significant rise in Psys and pronounced tachycardia (secondary response). Pretreatment with NG-nitro-l-arginine methyl ester reduced the NK-induced systemic vasodilatation, indicating that the effect is mediated through increased NO synthesis. The tachycardia associated with the late primary and secondary response to BK was abolished with propranolol and the systemic vasodilatation produced in the primary phase was also significantly attenuated by pretreatment, indicating that the responses are caused, at least in part, by release of cathecholamines and subsequent stimulation of β-adrenergic receptors. In contrast, the pulmonary circulation was relatively unresponsive to BK.
Genetic AT1Areceptor deficiency attenuates cold-induced hypertension
Authors: Sun Zhongjie , Wang Xiuqing , Wood Charles E. , Cade J. Robert
Published: October 2004

The aim of this study was to test our hypotheses that AT1Areceptors play a role in the pathogenesis of cold-induced hypertension (CIH) and in the cold-induced increase in drinking responses to ANG II. Two groups of wild-type (WT) and two groups of AT1Areceptor gene knockout (AT1A-KO) mice were used (6/group). Blood pressures (BP) of the four groups were similar during the control period at room temperature (25°C). After the control period, one group of WT and one group of AT1A-KO mice were exposed to cold (5°C), while the remaining groups were kept at 25°C. BP of the cold-exposed WT group elevated significantly within 1 wk of exposure to cold and increased gradually to a maximum level by week 5. However, there was only a slight increase in BP of the cold-exposed AT1A-KO group. The maximal cold-induced increase in BP (ΔBP) is significantly less in AT1A-KO group (11 ± 3 mmHg) than in WT group (49 ± 6 mmHg), indicating that AT1Areceptor deficiency attenuates cold-induced elevation of BP. Interestingly, both WT and AT1A-KO mice developed cardiac and renal hypertrophy to the same extent. AT1A-KO caused a significant increase in urine and plasma levels of nitric oxide (NO), indicating that the renin-angiotensin system inhibits NO formation probably via AT1Areceptors. Cold exposure inhibited endothelial NO synthase protein expressions and decreased urine and plasma levels of NO, which may be mediated partially by AT1Areceptors. AT1A-KO completely abolished the cold-induced increase in drinking responses to ANG II. We conclude that 1) AT1Areceptors play an essential role in the pathogenesis of CIH but not cardiac hypertrophy; 2) the role of AT1Areceptors in CIH may be mediated partially by its inhibitory effect on the NO system; and 3) cold-induced increase in drinking response to ANG II is mediated by AT1Areceptors.
Hypoxia induces major effects on cell cycle kinetics and protein expression inDrosophila melanogasterembryos
Authors: Douglas R. M. , Farahani R. , Morcillo P. , Kanaan A. , Xu T. , Haddad G. G.
Published: October 2004

Hypoxia induces a stereotypic response in Drosophila melanogaster embryos: depending on the time of hypoxia, embryos arrest cell cycle activity either at metaphase or just before S phase. To understand the mechanisms underlying hypoxia-induced arrest, two kinds of experiments were conducted. First, embryos carrying a kinesin-green fluorescent protein construct, which permits in vivo confocal microscopic visualization of the cell cycle, showed a dose-response relation between O2level and cell cycle length. For example, mild hypoxia (Po2∼55 Torr) had no apparent effect on cell cycle length, whereas severe hypoxia (Po2∼25–35 Torr) or anoxia (Po2= 0 Torr) arrested the cell cycle. Second, we utilized Drosophila embryos carrying a heat shock promoter driving the string ( cdc25) gene (HS-STG3), which permits synchronization of embryos before the start of mitosis. Under conditions of anoxia, we induced a stabilization or an increase in the expression of several G1/S (e.g., dE2F1, RBF2) and G2/M (e.g., cyclin A, cyclin B, dWee1) proteins. This study suggests that, in fruit fly embryos, 1) there is a dose-dependent relationship between cell cycle length and O2levels in fruit fly embryos, and 2) stabilized cyclin A and E2F1 are likely to be the mediators of hypoxia-induced arrest at metaphase and pre-S phase.
Blood pressure power within frequency range ∼0.4 Hz in rat conforms to self-similar scaling following spinal cord transection
Authors: Randall David C. , Baldridge Bobby R. , Zimmerman Ethan E. , Carroll Jonathan J. , Speakman Richard O. , Brown David R. , Taylor Robert F. , Patwardhan Abhijit , Burgess Don E.
Published: October 2004

This study quantified the effect of interrupting the descending input to the sympathetic preganglionic neurons on the dynamic behavior of arterial blood pressure (BP) in the unanesthetized rat. BP was recorded for ∼4-h intervals in six rats in the neurally intact state and in the same animals after complete spinal cord transection (SCT) between T4 and T5. In the intact state, power within the frequency range of 0.35–0.45 Hz was 1.53 ± 0.38 mmHg2/Hz (mean ± SD by fast Fourier transform). One week after SCT, power within this range decreased significantly ( P < 0.05) to 0.43 ± 0.62 mmHg2/Hz. To test for self-similarity before and after SCT, we analyzed data using a wavelet (i.e., functionally, a digital bandpass filter) tuned to be maximally sensitive to fluctuations with periods of ∼2, 4, 8, 16, 32, or 64 s. In the control state, all fluctuations with periods of ≥4 s conformed to a “self-similar” (i.e., fractal) distribution. In marked contrast, the oscillations with a period of ∼2 s (i.e., ∼0.4 Hz) were significantly set apart from those at lower frequencies. One day and seven days after the complete SCT, however, the BP fluctuations at ∼0.4 Hz now also conformed to the same self-similar behavior characteristic of the lower frequencies. We conclude that 1) an intact sympathetic nervous system endows that portion of the power spectrum centered around ∼0.4 Hz with properties (e.g., a periodicity) that differ significantly from the self-similar behavior that characterizes the lower frequencies and 2) even within the relatively high frequency range at 0.4 Hz self-similarity is the “default” condition after sympathetic influences have been eliminated.
Attenuation of T-lymphocyte demargination and adhesion molecule expression in response to moderate exercise in physically fit individuals
Authors: Hong Suzi , Johnson Todd A. , Farag Noha H. , Guy Harold J. , Matthews Scott C. , Ziegler Michael G. , Mills Paul J.
Published: October 2004

The effects of physical fitness on leukocyte demargination and cellular adhesion molecule (CAM) responses to moderate exercise were examined. We assessed leukocyte subsets and CAM expression before, immediately after, and 10 min after a 20-min treadmill exercise at 65–70% peak oxygen consumption in fit vs. nonfit individuals. Physical fitness was determined by peak oxygen consumption during a treadmill test. Catecholamine levels were determined by radioenzymatic assay, and enumeration of cells and detection of CAM expression were assessed by flow cytometry. As expected, exercise led to significant increases in numbers of leukocyte subsets, regardless of fitness level ( P < 0.01). Values returned to near resting levels 10 min after exercise. More importantly, physically fit individuals showed attenuated responses to the moderate-exercise challenge in numbers of CD3+, CD4+, CD8+, memory (CD45RO+) CD4, and naive (CD45RA+62L+) CD4 and CD8 lymphocytes. Postexercise human leukocyte antigen-DR absent memory CD4+ cell numbers were also lower in fit subjects. Increases in CD62L-expressing CD4+ and CD8+ lymphocytes and CD11a- expressing lymphocytes after exercise were also attenuated in fit individuals compared with nonfit individuals ( P < 0.05). Catecholamine levels increased to a similar extent ( P < 0.01) in both fitness groups. The findings suggest that physical fitness attenuates demargination of selected lymphocyte subsets in response to moderate exercise. Although the differences in plasma catecholamine responses were not significant between the groups, a possible mediating role of the sympathetic system remains to be further investigated. Being physically fit may offset exaggerated immune cell responses to stress.
Long-term bed rest-induced reductions in stroke volume during rest and exercise: cardiac dysfunction vs. volume depletion
Authors: Spaak Jonas , Montmerle Stéphanie , Sundblad Patrik , Linnarsson Dag
Published: October 2004

Long-term head-down-tilt bed rest (HDT) causes cardiovascular deconditioning, attributed to reflex dysfunctions, plasma volume reduction, or cardiac impairments. Our objective with the present study was to evaluate the functional importance and relative contribution of these during rest and exercise in supine and upright postures. We studied six subjects before (baseline), during [ days 60 (D60) and 113 (D113)], and after [recovery days 0 (R0), 3 (R3), and 15 (R15)] 120 days of −6° HDT. We determined cardiac output, stroke volume (SV), mean arterial pressure, and heart rate during rest and exercise in supine and upright postures. Cardiac output and SV decreased significantly in all four conditions, but the time courses differed for rest and exercise. Upright resting SV was decreased by 24 ± 9% at D60 compared with baseline but had recovered already at R3. Supine exercise SV decreased more slowly (by 5 ± 8% at D60 and by 18 ± 4% at D113) and recovered more slowly after HDT termination. Steady-state mean arterial pressure showed no changes. Heart rate had increased by 18 ± 4% at D60 and had recovered partially at R3. Our data indicate that long-term HDT causes both a rapid, preload-dependent reduction in SV, most evident during rest in the upright position, and a more slowly developing cardiac dysfunction, most evident during supine exercise. However, the ability to maintain blood pressure and to perform sustained low levels of dynamic exercise is not influenced by HDT.
Acyl-CoenzymeA (CoA):Cholesterol Acyltransferase Inhibition in Rat and Human Aortic Smooth Muscle Cells Is Nontoxic and Retards Foam Cell Formation
Authors: Rong James X. , Kusunoki Jun , Oelkers Peter , Sturley Stephen L. , Fisher Edward A.
Published: October 2004

Objective—

Studies in vitro and in vivo of macrophage foam cells have shown evidence of cytotoxicity after acyl-CoA:cholesterol acyltransferase (ACAT) inhibition. Foam cells of smooth muscle origin are also found in human and animal atherosclerotic lesions.

Methods and Results—

To study whether cytotoxicity from ACAT inhibition is independent of cell type, we first established a protocol to conveniently induce aortic smooth muscle foam cell formation using cholesterol–cyclodextrin complexes (CCC). Rat aortic smooth muscle cells (ASMCs) treated for 48 hours with CCC (20 μg/mL) became foam cells by morphological (oil-red-O staining) and biochemical (≈1200% and ≈180% increase in cellular esterified and free cholesterol, respectively) criteria. ACAT activity increased 500% (
P
<0.01 versus untreated). Similar results were obtained in human ASMC, but ACAT activity increased to an even greater extent (3200%;
P
<0.01 versus untreated). Western blots indicated that CCC treatment increased human (to 380±20% of untreated,
P
<0.001), but not rat, ACAT protein expression. ACAT inhibition by Fujirebio compound F1394 suppressed CCC-induced foam cell formation in rat and human ASMC, but, notably, did not induce significant cytotoxicity.

Conclusion—

ASMC might be more resistant to FC-induced adverse effects than are macrophages.
Association of Lipoprotein-Associated Phospholipase A
2
Mass and Activity With Calcified Coronary Plaque in Young Adults
Authors: Iribarren Carlos , Gross Myron D. , Darbinian Jeanne A. , Jacobs David R. , Sidney Stephen , Loria Catherine M.
Published: October 2004

Objective—

To examine the association of lipoprotein-associated phospholipase A
2
(Lp-PLA
2
) mass and activity with calcified coronary plaque in young adults.

Methods and Results—

Nested case-control study among CARDIA participants at the year 15 examination (2000 to 2001, 33 to 45 years old). Cases (n=266) were those with and controls (n=266) those without evidence of calcified coronary plaque by computed tomography matched 1:1 on sex and race. Lp-PLA
2
mass and activity were significantly higher in cases (296±101 ng/mL and 36.4±12.3 nmol/mL per minute) than in controls (267±80 ng/mL and 32.9±11.8 nmol/mL per minute). In age-adjusted conditional logistic regression, the odds ratio (OR) of calcified coronary plaque per 1 standard deviation (SD) increment was 1.40 (95% CI, 1.17 to 1.67) and 1.39 (95% CI, 1.14 to 1.70) for Lp-PLA
2
mass and activity, respectively. After adjusting for multiple covariates including low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides, and C-reactive protein, a statistically significant association remained for Lp-PLA
2
mass (OR, 1.28; 95% CI, 1.03 to 1.60) but not for activity (OR, 1.09; 95% CI, 0.84 to 1.42). No evidence was found for interaction between Lp-PLA
2
mass or activity with LDL-C as predictors of calcified coronary plaque.

Conclusion—

An independent association of Lp-PLA
2
mass with calcified coronary plaque existed in young adults. Therefore, Lp-PLA
2
mass may be a useful marker of subclinical cardiovascular risk.
ACAT1 Deficiency Disrupts Cholesterol Efflux and Alters Cellular Morphology in Macrophages
Authors: Dove Dwayne E. , Su Yan Ru , Zhang Wenwu , Jerome W. Gray , Swift Larry L. , Linton MacRae F. , Fazio Sergio
Published: October 2004

Objective—

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) converts intracellular free cholesterol (FC) into cholesteryl esters (CE) for storage in lipid droplets. Recent studies in our laboratory have shown that the deletion of the macrophage ACAT1 gene results in apoptosis and increased atherosclerotic lesion area in the aortas of hyperlipidemic mice. The objective of the current study was to elucidate the mechanism of the increased atherosclerosis.

Methods and Results—

CE storage and FC efflux were studied in ACAT1
(−/−)
peritoneal macrophages that were treated with acetylated low-density lipoprotein (acLDL). Our results show that efflux of cellular cholesterol was reduced by 25% in ACAT1-deficient cells compared with wild-type controls. This decrease occurred despite the upregulated expression of ABCA1, an important mediator of cholesterol efflux. In contrast, ACAT1 deficiency increased efflux of the cholesterol derived from acLDL by 32%. ACAT1-deficient macrophages also showed a 26% increase in the accumulation of FC derived from acLDL, which was associated with a 75% increase in the number of intracellular vesicles.

Conclusions—

Together, these data show that macrophage ACAT1 influences the efflux of both cellular and lipoprotein-derived cholesterol and propose a pathway for the pro-atherogenic transformation of ACAT1
(−/−)
macrophages.
Oral Flavonoid Supplementation Attenuates Atherosclerosis Development in Apolipoprotein E–Deficient Mice
Authors: Hishikawa Keiichi , Nakaki Toshio , Fujita Toshiro
Published: October 2004

Objective—

Caffeic acid phenethyl ester (CAPE), a natural flavonoid, specifically blocks activation of nuclear factor-κB (NF-κB). We examined the effects of oral CAPE supplementation on atherogenesis in apolipoprotein E–deficient (apoE−/−) mice.

Methods and Results—

Ten-week-old male apoE−/− mice were supplemented orally with CAPE (30 mg/kg body weight) for 12 weeks. At the end of administration, atherosclerosis progression, NF-κB activity, gene expression profiling by microarray analysis, and oxidative stress were studied. Treatment of apoE−/− mice with CAPE significantly reduced aortic atherosclerosis, NF-κB activity, and expression of NF-κB–related genes in the aorta. Moreover, expression of other gene clusters such as basic transcription factors, growth factors, cytokines, cell adhesion proteins, and extracellular matrix were also significantly reduced by treatment with CAPE. Plasma isoprostane level in apoE−/− mice was also significantly reduced by CAPE.

Conclusion—

In apoE−/− mice, oral CAPE supplementation attenuates the atherosclerotic process. This may be attributable to direct inhibition of NF-κB in the lesion and reduction of systemic oxidative stress.
Angiotensin II–Induced Protein Kinase D Activation Is Regulated by Protein Kinase Cδ and Mediated via the Angiotensin II Type 1 Receptor in Vascular Smooth Muscle Cells
Authors: Tan Mingqi , Xu Xuemin , Ohba Motoi , Cui Mei-Zhen
Published: October 2004

Objective—

Angiotensin II (Ang II), through its specific signaling cascades, exerts multiple effects on vascular smooth muscle cells (SMCs). It has been shown that Ang II stimulates activation of protein kinase D (PKD), a member of a new class of serine–threonine kinases. However, little is known regarding the upstream cascade of the intracellular signaling that leads to PKD activation. In the present study, we investigated upstream molecules that mediate Ang II–induced PKD activation in SMCs.

Methods and Results—

Protein kinase C (PKC) inhibitors completely block Ang II–induced PKD activation, and pretreatment with phorbol 12,13-dibutyrate downregulates Ang II–induced PKD activation, indicating that classical or novel isoforms of PKC mediate Ang II–induced PKD activation. Furthermore, the finding that rottlerin, a PKCδ-specific inhibitor, blocks PKD activation suggests that PKCδ, a member of novel PKCs, mediates Ang II–induced PKD activation. By using dominant-negative approaches, our results demonstrate that expression of the dominant-negative PKCδ, but neither the dominant-negative form of PKCε nor PKCζ, inhibits PKD activation. These results further substantiate the finding that Ang II–induced PKD activation is mediated by PKCδ. Moreover, using selective Ang II receptor antagonists, our data show that the Ang II type 1 (AT1) receptor but not the AT2 mediates Ang II–stimulated PKD activation.

Conclusions—

This study reveals for the first time that Ang II–induced PKD activation is mediated via AT1 and regulated by PKCδ in living cells. These data may provide new insights into molecular mechanisms involved in Ang II–induced physiological and pathological events.
Vitamin D, Shedding Light on the Development of Disease in Peripheral Arteries
Authors: Norman P.E. , Powell J.T.
Published: October 2004

Vitamin D is generally associated with calcium metabolism, especially in the context of uptake in the intestine and the formation and maintenance of bone. However, vitamin D influences a wide range of metabolic systems through both genomic and nongenomic pathways that have an impact on the properties of peripheral arteries. The genomic effects have wide importance for angiogenesis, elastogenesis, and immunomodulation; the nongenomic effects have mainly been observed in the presence of hypertension. Although some vitamin D is essential for cardiovascular health, excess may have detrimental effects, particularly on elastogenesis and inflammation of the arterial wall. Vitamin D is likely to have a role in the paradoxical association between arterial calcification and osteoporosis. This review explores the relationship between vitamin D and a range of physiological and pathological processes relevant to peripheral arteries.
Uterine Spiral Artery Remodeling Involves Endothelial Apoptosis Induced by Extravillous Trophoblasts Through Fas/FasL Interactions
Authors: Ashton Sandra V. , Whitley Guy St. J. , Dash Philip R. , Wareing Mark , Crocker Ian P. , Baker Philip N. , Cartwright Judith E.
Published: October 2004

Objective—Invasion of uterine spiral arteries by extravillous trophoblasts in the first trimester of pregnancy results in loss of endothelial and musculoelastic layers. This remodeling is crucial for an adequate blood supply to the fetus with a failure to remodel implicated in the etiology of the hypertensive disorder preeclampsia. The mechanism by which trophoblasts induce this key process is unknown. This study gives the first insights into the potential mechanisms involved.

Methods and Results—Spiral arteries were dissected from nonplacental bed biopsies obtained at Caesarean section, and a novel model was used to mimic in vivo events. Arteries were cultured with trophoblasts in the lumen, and apoptotic changes in the endothelial layer were detected after 20 hours, leading to loss of endothelium by 96 hours. In vitro, coculture experiments showed that trophoblasts stimulated apoptosis of primary decidual endothelial cells and an endothelial cell line. This was blocked by caspase inhibition and NOK2, a FasL blocking antibody. NOK2 also abrogated trophoblast-induced endothelial apoptosis in the vessel model.

Conclusions—Extravillous trophoblast induction of endothelial apoptosis is a possible mechanism by which the endothelium is removed, and vascular remodeling may occur in uterine spiral arteries. Fas/FasL interactions have an important role in trophoblast-induced endothelial apoptosis.
Reduced Macrophage Apoptosis Is Associated With Accelerated Atherosclerosis in Low-Density Lipoprotein Receptor-Null Mice
Authors: Liu June , Thewke Douglas P. , Su Yan Ru , Linton MacRae F. , Fazio Sergio , Sinensky Michael S.
Published: October 2004

Objective—The majority of apoptotic cells in atherosclerotic lesions are macrophages. However, the pathogenic role of macrophage apoptosis in the development of atherosclerosis remains unclear. Elevated expression of Bax, one of the pivotal proapoptotic proteins of the Bcl-2 family, has been found in human atherosclerotic plaques. Activation of Bax also occurs in free cholesterol-loaded and oxysterol-treated mouse macrophages. In this study, we examined the effect of Bax deficiency in bone marrow-derived leukocytes on the development of atherosclerosis in low-density lipoprotein receptor-null (LDLR−/−) mice.

Methods and Results—Fourteen 8-week-old male LDLR−/− mice were lethally irradiated and reconstituted with either wild-type (WT) C57BL6 or Bax-null (Bax−/−) bone marrow. Three weeks later, the mice were challenged with a Western diet for 10 weeks. No differences were found in the plasma cholesterol level between the WT and Bax−/− group. However, quantitation of cross sections from proximal aorta revealed a 49.2% increase (P=0.0259) in the mean lesion area of the Bax−/− group compared with the WT group. A 53% decrease in apoptotic macrophages in the Bax−/− group was found by TUNEL staining (P<0.05).

Conclusions—The reduction of apoptotic activity in macrophages stimulates atherosclerosis in LDLR−/− mice, which is consistent with the hypothesis that macrophage apoptosis suppresses the development of atherosclerosis.
Chronic Treatment With Allopurinol Boosts Survival and Cardiac Contractility in Murine Postischemic Cardiomyopathy
Authors: Stull Linda B. , Leppo Michelle K. , Szweda Luke , Gao Wei Dong , Marbán Eduardo
Published: October 2004

Oxidative stress is a hallmark of systemic illnesses, including heart failure. Nevertheless, the overall importance of radical production in the heart remains conjectural; is it merely a marker of illness, or can intervention alter the progression of disease? This question was addressed by blocking xanthine oxidase (XO), a superoxide-generating enzyme that is upregulated in animal models of heart failure. In a randomized prospective trial design, we administered the XO inhibitor allopurinol orally to mice that had undergone massive myocardial infarction (MI). Cardiac XO activity was elevated in untreated mice after MI; allopurinol suppressed the XO activity to levels comparable to those in sham-operated mice. Eighty-one percent of untreated mice died of advanced heart failure over 2 to 4 weeks of follow-up. Survival doubled in the allopurinol-treated mice, whereas cardiac contractile function (both in vivo and in isolated muscle) was markedly improved. Response to isoproterenol was restored to near-normal levels in the allopurinol group but was attenuated in untreated mice. Oxidative modifications to proteins were prevented in the allopurinol-treated mice. Our findings indicate that targeted blockade of just one source of oxidants, XO, impacts dramatically on the progression of postischemic cardiomyopathy in mice and prevents oxidative protein modifications.
Extent and Distribution of White Matter Hyperintensities in Stroke Patients
Authors: Wen Wei , Sachdev Perminder S.
Published: October 2004

Background and Purpose—White matter hyperintensities (WMHs) on T2-weighted MRI are common in stroke patients and healthy elderly individuals. The detailed anatomical distribution of these lesions in stroke patients has not been examined.

Methods—A total of 112 stroke or transient ischemic attack patients and 87 matched control subjects from the Sydney Stroke Study underwent MRI scans that included a T2-weighted fluid-attenuated inversion recovery (FLAIR) sequence. WMHs were delineated from each FLAIR MRI by an automated method. Region of interest and voxel-wise statistical parametric mapping approaches were applied to examine the volume, distribution, and severity of WMHs of the patient and control groups, and subgroups with large or lacunar infarcts.

Results—Stroke subjects had significantly more WMHs than controls in all brain regions except the occipital lobe and in all arterial territories except the anterior callosal and anterior medial lenticulostriate. In the frontotemporal regions, average WMH volumes in patients were >3.5× those in controls. The total number of discrete WMHs was not different in the 2 groups, but stroke patients had more large (>20 mm) and high-intensity lesions. Subjects with lacunar infarcts had more WMHs than those with large infarcts, who, in turn, had more WMHs than control subjects. Lacunar infarction subjects had more WMHs than subjects with large thromboembolic or cardioembolic strokes. Those with anterior arterial territory infarction had more WMHs in the frontal regions. Subjects with single or multiple lacunes did not differ in volumes of WMHs.

Conclusions—Stroke patients have significantly more WMHs in nearly every brain region than healthy controls. Those with lacunar infarcts are particularly affected. WMHs represent a significant proportion of the ischemic lesion burden in stroke and transient ischemic attack patients.
High Incidence of Neuroendocrine Dysfunction in Long-Term Survivors of Aneurysmal Subarachnoid Hemorrhage
Authors: Dimopoulou Ioanna , Kouyialis Andreas T. , Tzanella Marinella , Armaganidis Apostolos , Thalassinos Nikolaos , Sakas Damianos E. , Tsagarakis Stylianos
Published: October 2004

Background and Purpose—

To investigate the incidence, pattern, and magnitude of neuroendocrine changes in long-term survivors of aneurysmal subarachnoid hemorrhage (SAH).

Methods—

Thirty patients (16 women) with a mean age of 50±13 years underwent endocrine assessment between 12 and 24 months after aneurysmal SAH. SAH severity was graded clinically by the Hunt & Hess scale (median, II) and radiologically by the Fisher classification (median, II). Patients underwent measurement of basal hormone levels and dynamic assessment by the low-dose (1 μg) corticotropin stimulation test. Functional outcome was examined concurrently with endocrine testing by the modified Rankin Scale and the Barthel Index.

Results—

Of the 30 patients tested, 14 patients (47%) showed isolated or combined endocrine abnormalities. These included low insulin-like growth factor 1 levels compatible with growth hormone deficiency in 37%, hypogonadism in 13%, and cortisol hyporesponsiveness to the low-dose corticotropin stimulation test in 10%; thyroid dysfunction in the form of subclinical hypothyroidism was observed in 7% of patients. Median modified Rankin Scale and Barthel Index at the time of endocrine testing were 1 and 100, respectively. There was no correlation between the presence of endocrine dysfunction and SAH severity indices or functional outcome scores.

Conclusions—

Long-term survivors of aneurysmal SAH frequently exhibit endocrine changes, with growth hormone and gonadal deficiencies predominating. Thus, screening of pituitary function is recommended in patients surviving SAH. The relationship between late hormonal alterations and functional outcome in patients with SAH warrants further study.
IFN-γ gene polymorphisms associate with development of EBV+ lymphoproliferative disease in hu PBL-SCID mice
Authors: Dierksheide Julie E. , Baiocchi Robert A. , Ferketich Amy K. , Roychowdhury Sameek , Pelletier Ronald P. , Eisenbeis Charles F. , Caligiuri Michael A. , VanBuskirk Anne M.
Published: October 2004

Posttransplantation lymphoproliferative disorder (PTLD) is a devastating post-transplantation complication often associated with Epstein-Barr virus (EBV). Although the type and length of immunosuppression are risk factors, a patient's inherent immune capacity also likely contributes to this disorder. This report uses severe-combined immunodeficient mice given injections of human peripheral blood leukocytes (hu PBL-SCID [Severe Combined Immunodeficient] mice) to test the hypothesis that cytokine genotype associates with the development of EBV-associated lymphoproliferative disease (LPD). We observed that the A/A (adenosine/adenosine) genotype for base + 874 of the interferon γ (IFN-γ) gene was significantly more prevalent in PBLs producing rapid, high-penetrance LPD in hu PBL-SCID mice, compared to PBLs producing late, lowpenetrance LPD or no LPD. In examining the relationship between genotype and cytolytic T-lymphocyte (CTL) function, transforming growth factor β (TGF-β) inhibited restimulation of CTLs in PBLs with adenosine at IFNG base + 874, but not in PBLs homozygous for thymidine. Importantly, neutralization of TGF-β in hu PBL-SCID mice injected with A/A genotype PBLs resulted in reduced LPD development and expanded human CD8+ cells. Thus, our data show that TGF-β may promote tumor development by inhibiting CTL restimulation and expansion. Further, our data indicate that IFNG genotype may provide valuable information for both identifying transplant recipients at greater risk for PTLD and developing preventive and curative strategies.
Smad7 alters cell fate decisions of human hematopoietic repopulating cells
Authors: Chadwick Kristin , Shojaei Farbod , Gallacher Lisa , Bhatia Mickie
Published: October 2004

Intracellular Smad proteins mediate signal transduction of the transforming growth factor-β (TGF-β) superfamily that play pleiotropic roles in hematopoietic development, suggesting that intracellular Smad proteins may play key roles in hematopoietic regulation. Although inhibitory Smad7, which negatively regulates TGF-β signaling, has been implicated in the development of mature hematopoietic cells, a role for Smad7 in regulating more primitive hematopoietic cells has yet to be examined. Here, Smad7 was overexpressed in primary human severe combined immunodeficient (SCID) repopulating cells (SRCs), representing a common myeloid/lymphoid precursor cell with the functional capacity to repopulate the bone marrow of nonobese diabetic (NOD)/SCID recipient mice. Retroviral transduction of Smad7 into human umbilical cord blood (CB)-SRCs caused a shift from lymphoid dominant engraftment toward increased myeloid contribution, and increased the myeloid-committed clonogenic progenitor frequency in reconstituted mice. Neither myeloid nor B-lymphoid lineage developmental stages were compromised by Smad7 overexpression, suggesting Smad7 regulates cell fate commitment decisions of myeloid/lymphoid precursors by augmenting myeloid differentiation at the expense of lymphoid commitment. In addition, global gene expression analysis using microarray was used to identify potential target genes regulated by Smad7 in primitive hematopoietic cells that may control this process. Our study demonstrates a novel and unexpected role for Smad7 in modulating the cell fate decisions of primary multipotent human repopulating cells and establishes a role for Smad7 in the development of primitive human hematopoietic cells.
Preconditioning of NOD mice with anti-CD8 mAb and costimulatory blockade enhances chimerism and tolerance and prevents diabetes, while depletion of αβ-TCR+ and CD4+ cells negates the effect
Authors: Ildstad Suzanne T. , Chilton Paula M. , Xu Hong , Domenick Michele A. , Ray Mukunda B.
Published: October 2004

Bone marrow transplantation blocks diabetes pathogenesis and reverses autoimmunity in nonobese diabetic (NOD) mice. However, there is a greater barrier to engraftment in the context of autoimmunity. In the present study, we characterized which recipient cells influence engraftment in prediabetic NOD mice, with the goal to replace myelotoxic conditioning with antigen-specific deletion of reactive host cells. Preconditioning of NOD mice with anti-CD8 and anti-CD154 monoclonal antibodies (mAbs) synergistically enhanced engraftment and significantly reduced the minimum total body irradiation (TBI) dose for engraftment. Strikingly, preconditioning with anti-CD4 mAb significantly impaired engraftment, negating the beneficial effect of anti-CD8, and resulted in a requirement for more TBI-based conditioning compared with controls conditioned with TBI alone. Similarly, more TBI was required when anti–T-cell receptor β (TCRβ) mAb was administered as preconditioning. The addition of anti-CD152 to CD154 preconditioning abrogated the engraftment-enhancing effect of anti-CD154. Taken together, these data indicate a role for CD4+ regulatory T cells in vivo which require signaling via CD152 in the induction of chimerism and tolerance in NOD recipients. Notably, disease prevention and reversal of autoimmunity was absolutely correlated with the establishment of chimerism. These studies have important implications for the design of novel clinical approaches to treat type 1 diabetes.
Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival
Authors: Kim Kyu-Tae , Baird Kristin , Ahn Joon-Young , Meltzer Paul , Lilly Michael , Levis Mark , Small Donald
Published: October 2004

Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) play an important role in leukemogenesis, and their presence is associated with poor prognosis in acute myeloid leukemia (AML). To better understand FLT3 signaling in leukemogenesis, we have examined the changes in gene expression induced by FLT3/ITD or constitutively activated wild-type FLT3 expression. Microarrays were used with RNA harvested before and after inhibition of FLT3 signaling. Pim-1 was found to be one of the most significantly down-regulated genes upon FLT3 inhibition. Pim-1 is a proto-oncogene and is known to be up-regulated by signal transducer and activator of transcription 5 (STAT5), which itself is a downstream target of FLT3 signaling. Quantitative polymerase chain reaction (QPCR) confirmed the microarray results and demonstrated approximately 10-fold decreases in Pim-1 expression in response to FLT3 inhibition. Pim-1 protein also decreased rapidly in parallel with decreasing autophosphorylation activity of FLT3. Enforced expression of either the 44-kDa or 33-kDa Pim-1 isotypes resulted in increased resistance to FLT3 inhibition-mediated cytotoxicity and apoptosis. In contrast, expression of a dominant-negative Pim-1 construct accelerated cytotoxicity in response to FLT3 inhibition and inhibited colony growth of FLT3/ITD-transformed BaF3 cells. These findings demonstrate that constitutively activated FLT3 signaling up-regulates Pim-1 expression in leukemia cells. This up-regulation contributes to the proliferative and antiapoptotic pathways induced by FLT3 signaling. (Blood. 2005;105: 1759-1767)
A STAT5 modifier locus on murine chromosome 7 modulates engraftment of hematopoietic stem cells during steady-state hematopoiesis
Authors: Couldrey Christine , Bradley Heath L. , Bunting Kevin D.
Published: October 2004

Homologous disruption of expression of signal transducer and activator of transcription 5a (STAT5a) and STAT5b (STAT5ab–/–) in mice results in hematopoietic stem cells (HSCs) that can engraft irradiated hosts alone but are noncompetitive against wild-type HSCs. To explore mechanisms for this phenotype, we crossed the STAT5 mutations onto an HW80 background congenic to the original C57BL/6 that differs in a small chromosome 7 genomic locus. We previously demonstrated that C57BL/6 or HW80 background STAT5ab–/– bone marrow (BM) cells showed equal repopulating function either competitively or noncompetitively in irradiated hosts. However, one intraperitoneal injection of wild-type green fluorescent protein (GFP) transgenic BM cells into unconditioned newborn STAT5ab–/– recipients of either background was sufficient for high-level donor engraftment. Furthermore, haploinsufficiency of STAT5 (STAT5ab+/–) allowed improved engraftment over wild-type recipients, indicating a dose-dependent requirement for STAT5 activation. In reciprocal experiments, STAT5ab–/– BM was transplanted into nonirradiated W/Wv hosts. In these mice, C57BL/6 STAT5ab–/– BM cells were 10-fold more defective in long-term engraftment than control wild-type BM cells and HW80 STAT5ab–/– BM cells were 5- to 10-fold more defective than C57BL/6 STAT5ab–/– BM cells. Therefore, we conclude that STAT5 plays a critical role during steady-state HSC engraftment and a chromosome 7 modifier locus regulates this activity.
Novel chromosomal imbalances in mantle cell lymphoma detected by genome-wide array-based comparative genomic hybridization
Authors: Schraders Margit , Pfundt Rolph , Straatman Huub M. P. , Janssen Irene M. , van Kessel Ad Geurts , Schoenmakers Eric F. P. M. , van Krieken Johan H. J. M. , Groenen Patricia J. T. A.
Published: October 2004

Mantle cell lymphoma (MCL) is an aggressive, highly proliferative B-cell non-Hodgkin lymphoma, characterized by the specific t(11;14)(q13;q32) translocation. It is well established that this translocation alone is not sufficient to promote MCL development, but that additional genetic changes are essential for malignant transformation. We have identified such additional tumorigenic triggers in MCL tumors, by applying genome-wide array-based comparative genomic hybridization with an 800-kilobase (kb) resolution. This strategy, combined with a newly developed statistical approach, enabled us to confirm previously reported genomic alterations such as loss of 1p, 6q, 11q, 13q and gain of 3q and 8q, but it also facilitated the detection of novel recurrent genomic imbalances, such as gain of 4p12-13 and loss of 20p12.1-12.3, 20q12-13.2, 22q12.1-12.3, and 22q13.31-13.32. Genomic hotspot detection allowed for the identification of small genomic intervals that are frequently affected (57%-93%), resulting in interesting positional candidate genes such as KITLG, GPC5, and ING1. Finally, by assessing multiple biopsies from the same patient, we show that seemingly stable genomes do show subtle genomic changes over time. The follow-up of multiple biopsies of patients with MCL by high-resolution genomic profiling is expected to provide us with new clues regarding the relation between clinical outcome and in vivo cytogenetic evolution. (Blood. 2005;105:1686-1693)