bluuebunny/crossref_metadata_2025 huggingface dataset:
Transgenic Myocardial Overexpression of Fibroblast Growth Factor-1 Increases Coronary Artery Density and Branching
Authors: Fernandez Borja , Buehler Alexandra , Wolfram Swen , Kostin Sawa , Espanion Gudrun , Franz Wolfgang M. , Niemann Heiner , Doevendans Pieter A. , Schaper Wolfgang , Zimmermann René
Published: June 2012

Abstract
—Fibroblast growth factor (FGF)-1 plays important roles during myocardial and coronary morphogenesis. FGF-1 is also involved in the physiological response of the adult heart against ischemia, which includes cardiomyocyte protection and vascular growth. In the present study, we have generated transgenic mice with specific myocardial overexpression of the gene. Transgene expression was verified by Northern blot, and increased FGF-1 protein content was assessed by Western blot and immunoconfocal microscopy. Anatomic, histomorphological, and ultrastructural analyses revealed no major morphological or developmental abnormalities of transgenic hearts. Capillary density was unaltered, whereas the density of coronary arteries, especially arterioles, was significantly increased, as was the number of branches of the main coronary arteries. In addition, the coronary flow was significantly enhanced in transgenic mice ex vivo. These differences in the anatomic pattern of the coronary vasculature are established during the second month of postnatal life. The present findings demonstrate an important role of FGF-1 in the differentiation and growth of the coronary system and suggest that it is a key regulatory molecule of the differentiation of the arterial system.
1α,25-Dihydroxyvitamin D
3
Inhibits Angiogenesis In Vitro and In Vivo
Authors: Mantell D. J. , Owens P. E. , Bundred N. J. , Mawer E. B. , Canfield A. E.
Published: June 2012

Abstract
—Modulation of angiogenesis is now a recognized strategy for the prevention and treatment of pathologies categorized by their reliance on a vascular supply. The purpose of this study was to evaluate the effect of 1α,25-dihydroxyvitamin D
3
[1,25(OH)
2
D
3
], the active metabolite of vitamin D
3
, on angiogenesis by using well-characterized in vitro and in vivo model systems. 1,25(OH)
2
D
3
(1×10
−
9
to 1×10
−
7
mol/L) significantly inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell sprouting and elongation in vitro in a dose-dependent manner and had a small, but significant, inhibitory effect on VEGF-induced endothelial cell proliferation. 1,25(OH)
2
D
3
also inhibited the formation of networks of elongated endothelial cells within 3D collagen gels. The addition of 1,25(OH)
2
D
3
to endothelial cell cultures containing sprouting elongated cells induced the regression of these cells, in the absence of any effect on cells present in the cobblestone monolayer. Analysis of nuclear morphology, DNA integrity, and enzymatic in situ labeling of apoptosis-induced strand breaks demonstrated that this regression was due to the induction of apoptosis specifically within the sprouting cell population. The effect of 1,25(OH)
2
D
3
on angiogenesis in vivo was investigated by using a model in which MCF-7 breast carcinoma cells, which had been induced to overexpress VEGF, were xenografted subcutaneously together with MDA-435S breast carcinoma cells into nude mice. Treatment with 1,25(OH)
2
D
3
(12.5 pmol/d for 8 weeks) produced tumors that were less well vascularized than tumors formed in mice treated with vehicle alone. These results highlight the potential use of 1,25(OH)
2
D
3
in both the prevention and regression of conditions characterized by pathological angiogenesis.
Protein Kinase C Activation Contributes to Microvascular Barrier Dysfunction in the Heart at Early Stages of Diabetes
Authors: Yuan Sarah Y. , Ustinova Elena E. , Wu Mack H. , Tinsley John H. , Xu Wenjuan , Korompai Ferenc L. , Taulman Amy C.
Published: June 2012

Abstract
—The functional disturbance of microvasculature is recognized as an initiating mechanism that underlies the development of various diabetic complications. Although a causal relationship between microvascular leakage and tissue damage has been well documented in diabetic kidneys and eyes, there is a lack of information regarding the barrier function of coronary exchange vessels in the disease state. The aim of the present study was to evaluate the permeability property of coronary microvessels during the early development of experimental diabetes with a focus on the protein kinase C (PKC)-dependent signaling mechanism. The apparent permeability coefficient of albumin (Pa) was measured in isolated and perfused porcine coronary venules. The administration of high concentrations of
d
-glucose induced a dose-dependent increase in the Pa value, which was prevented by blockage of PKC with its selective inhibitors bisindolylmaleimide and Goe 6976. More importantly, an elevated basal permeability to albumin was observed in coronary venules at the early onset of streptozotocin-induced diabetes. The hyperpermeability was corrected with bisindolylmaleimide and the selective PKCβ inhibitor hispidin. Concomitantly, protein kinase assay showed a high PKC activity in isolated diabetic venules. Immunoblot analysis of the diabetic heart revealed a significant subcellular translocation of PKCβII and PKCε from the cytosol to the membrane, indicating that the specific activity of these isoforms was preferentially elevated. The results suggest that endothelial barrier dysfunction attributed to the activation of PKC occurs at the coronary exchange vessels in early diabetes.
Inhibition of Matrix Metalloproteinases by Lung TIMP-1 Gene Transfer or Doxycycline Aggravates Pulmonary Hypertension in Rats
Authors: Vieillard-Baron Antoine , Frisdal Eric , Eddahibi Saadia , Deprez Isabelle , Baker Andrew H. , Newby Andrew C. , Berger Patrick , Levame Micheline , Raffestin Bernadette , Adnot Serge , d’Ortho Marie-Pia
Published: June 2012

Abstract
—Chronic hypoxic pulmonary hypertension (PH) results from persistent vasoconstriction, excess muscularization, and extracellular matrix remodeling of pulmonary arteries. The matrix metalloproteinases (MMPs) are a family of proteinases implicated in extracellular matrix turnover and hence in smooth muscle and endothelial cell migration and proliferation. Because MMP expression and activity are increased in PH, we designed the present study to investigate whether inhibition of lung MMPs in rats subjected to chronic hypoxia (CH) contributes to or protects against vascular remodeling and PH. To achieve lung MMP inhibition, rats exposed to 10% O
2
for 15 days were treated with either doxycycline (20 mg/kg per day by gavage starting 2 days before and continuing throughout the CH period) or a single dose of recombinant adenovirus (Ad) for the human tissue inhibitors of metalloproteinases-1 (hTIMP-1) gene (Ad.hTIMP-1, 10
8
plaque-forming units given intratracheally 2 days before CH initiation). Control groups either received no treatment or were treated with an adenovirus containing no gene in the expression cassette (Ad.Null). Efficacy of hTIMP-1 gene transfer was assessed both by ELISA on bronchoalveolar lavages and by hTIMP-1 immunofluorescence on lung sections. MMP inhibition in lungs was evaluated by in situ zymography and gelatinolytic activity assessment using [
3
H]gelatin. Rats treated with either doxycycline or Ad.hTIMP-1 had higher pulmonary artery pressure and right heart ventricular hypertrophy more severe than their respective controls. Worsening of PH was associated with increased muscularization and periadventitial collagen accumulation in distal arteries. In conclusion, our study provides compelling evidence that MMPs play a pivotal role in protecting against pulmonary artery remodeling.
Ionic Remodeling in the Heart
Authors: Nattel Stanley , Li Danshi
Published: June 2012

Abstract
—Heart disease has long been recognized to alter cardiac electrical function. Detailed studies of disease-induced remodeling of ionic transport processes that underlie ventricular electrophysiological alterations have been performed over the past 10 years, but our knowledge of atrial ionic remodeling is more limited and has emerged much more recently. The present review focuses on recent findings regarding ionic remodeling at the atrial level, particularly with respect to two conditions that promote atrial fibrillation (AF) in well-developed clinically relevant animal models: (1) sustained atrial tachycardia and (2) ventricular tachypacing–induced congestive heart failure. Complementary data from experimental models and from observations in atrial tissue samples from patients are examined critically and integrated. Consideration is also given to potential molecular mechanisms underlying remodeling, the relationship between atrial and ventricular ionic remodeling in response to similar stimuli, and the potential relevance of insights into ionic remodeling for understanding the pathophysiology of AF and developing improved therapeutic approaches.
Antiangiogenic Effect of Interleukin-10 in Ischemia-Induced Angiogenesis in Mice Hindlimb
Authors: Silvestre Jean-Sébastien , Mallat Ziad , Duriez Micheline , Tamarat Radia , Bureau Michel F. , Scherman Daniel , Duverger Nicolas , Branellec Didier , Tedgui Alain , Levy Bernard I.
Published: June 2012

Abstract
—Ischemia induces both hypoxia and inflammation that trigger angiogenesis. The inflammatory reaction is modulated by production of anti-inflammatory cytokines. This study examined the potential role of a major anti-inflammatory cytokine, interleukin (IL)–10, on angiogenesis in a model of surgically induced hindlimb ischemia. Ischemia was produced by artery femoral occlusion in both C57BL/6J IL-10
+/+
and IL-10
–/–
mice. After 28 days, angiogenesis was quantified by microangiography, capillary, and arteriole density measurement and laser Doppler perfusion imaging. The protein levels of IL-10 and vascular endothelial growth factor (VEGF) were determined by Western blot analysis in hindlimbs. IL-10 was markedly expressed in the ischemic hindlimb of IL-10
+/+
mice. Angiogenesis in the ischemic hindlimb was significantly increased in IL-10
–/–
compared with IL-10
+/+
mice. Indeed, angiographic data showed that vessel density in the ischemic leg was 10.2±0.1% and 5.7±0.4% in IL-10
–/–
and IL-10
+/+
mice, respectively (
P
<0.01). This corresponded to improved ischemic/nonischemic leg perfusion ratio by 1.4-fold in IL-10
–/–
mice compared with IL-10
+/+
mice (0.87±0.05 versus 0.63±0.01, respectively;
P
<0.01). Revascularization was associated with a 1.8-fold increase in tissue VEGF protein level in IL-10
–/–
mice compared with IL-10
+/+
mice (
P
<0.01). In vivo electrotransfer of murine IL-10 cDNA in IL-10
–/–
mice significantly inhibited both the angiogenic process and the rise in VEGF protein level observed in IL-10
–/–
mice. No changes in vessel density or VEGF content were observed in the nonischemic hindlimb. These findings underscore the antiangiogenic effect of IL-10 associated with the downregulation of VEGF expression and suggest a role for the inflammatory balance in the modulation of ischemia-induced angiogenesis.
Electrical Coupling Between Endothelial Cells and Smooth Muscle Cells in Hamster Feed Arteries
Authors: Emerson Geoffrey G. , Segal Steven S.
Published: June 2012

Abstract
—Endothelial cells (ECs) govern smooth muscle cell (SMC) tone via the release of paracrine factors (eg, NO and metabolites of arachidonic acid). We tested the hypothesis that ECs can promote SMC relaxation or contraction via direct electrical coupling. Vessels (resting diameter, 57±3 μm; length, 4 mm) were isolated, cannulated, and pressurized (75 mm Hg; 37°C). Two microelectrodes were used to simultaneously impale 2 cells (ECs or SMCs) in the vessel wall separated by 500 μm. Impalements of one EC and one SMC (n=26) displayed equivalent membrane potentials at rest, during spontaneous oscillations, and during hyperpolarization and vasodilation to acetylcholine. Injection of −0.8 nA into an EC caused hyperpolarization (≈5 mV) and relaxation of SMCs (dilation, ≈5 μm) along the vessel segment. In a reciprocal manner, +0.8 nA caused depolarization (≈2 mV) of SMCs with constriction (≈2 μm). Current injection into SMCs while recording from ECs produced similar results. We conclude that ECs and SMCs are electrically coupled to each other in these vessels, such that electrical signals conducted along the endothelium can be directly transmitted to the surrounding smooth muscle to evoke vasomotor responses.
Angiotensin II–Induced Ca
2+
Influx in Renal Afferent and Efferent Arterioles
Authors: Loutzenhiser Kathy , Loutzenhiser Rodger
Published: June 2012

Abstract
—Angiotensin II (Ang II)–induced Ca
2+
signaling was studied in isolated rat renal arterioles using fura-2. Ang II (10 nmol/L) caused a sustained elevation in [Ca
2+
]
i
, which was dependent on [Ca
2+
]
o
in both vessel types. This response was blocked by nifedipine in only the afferent arteriole. Using the Mn
2+
quench technique, we found that Ang II stimulates Ca
2+
influx in both vessels. Nifedipine blocked the Ang II–induced Ca
2+
influx in afferent arterioles but not in efferent arterioles. In contrast to Ang II, KCl-induced depolarization stimulated Ca
2+
influx in only the afferent arteriole. Cyclopiazonic acid (CPA, 30 μmol/L) was used to examine the presence of store-operated Ca
2+
entry in myocytes isolated from each arteriole. In efferent myocytes, CPA induced a sustained Ca
2+
increase that was dependent on [Ca
2+
]
o
and insensitive to nifedipine. This mechanism was absent in afferent myocytes. SKF 96365 inhibited Ang II–induced Ca
2+
entry in efferent arterioles and CPA-induced Ca
2+
entry in efferent myocytes over identical concentrations. Our findings thus indicate that Ang II activates differing Ca
2+
influx mechanisms in pre- and postglomerular arterioles. In the afferent arteriole, Ang II activates dihydropyridine-sensitive L-type Ca
2+
channels, presumably by membrane depolarization. In the efferent arteriole, Ang II appears to stimulate Ca
2+
entry via store-operated Ca
2+
influx.
Peroxisome Proliferator–Activated Receptor Activators Inhibit Lipopolysaccharide-Induced Tumor Necrosis Factor-α Expression in Neonatal Rat Cardiac Myocytes
Authors: Takano Hiroyuki , Nagai Toshio , Asakawa Masayuki , Toyozaki Tetsuya , Oka Toru , Komuro Issei , Saito Toshihiro , Masuda Yoshiaki
Published: June 2012

Abstract
—Peroxisome proliferator–activated receptors (PPARs) are transcription factors belonging to the nuclear receptor superfamily. Recently, PPAR activators have been shown to inhibit the production of proinflammatory cytokines in macrophages or vascular smooth muscle cells. It has been reported that tumor necrosis factor-α (TNF-α) expression is elevated in the failing heart and that TNF-α has a negative inotropic effect on cardiac myocytes. Therefore, we examined the effects of PPARα and PPARγ activators on expression of TNF-α in neonatal rat cardiac myocytes. Northern blot analysis revealed expression of PPARα and PPARγ mRNA in cardiac myocytes. Immunofluorescent staining demonstrated that both PPARα and PPARγ were expressed in the nuclei of cells. When cardiac myocytes were transfected with PPAR responsive element (PPRE)-luciferase reporter plasmid, both PPARα and PPARγ activators increased the promoter activity. Cardiomyocytes were stimulated with lipopolysaccharide (LPS), and the levels of TNF-α in the medium were measured by ELISA. After exposure to LPS, the levels of TNF-α significantly increased. However, pretreatment of myocytes with PPARα or PPARγ activators decreased LPS-induced expression of TNF-α in the medium. Both PPARα and PPARγ activators also inhibited LPS-induced increase in TNF-α mRNA in myocytes. In addition, electrophoretic mobility shift assays demonstrated that PPAR activators reduced LPS-induced nuclear factor-κB activation. These results suggest that both PPARα and PPARγ activators inhibit cardiac expression of TNF-α in part by antagonizing nuclear factor-κB activity and that treatment with PPAR activators may lead to improvement in congestive heart failure.
Dephosphorylation and Intracellular Redistribution of Ventricular Connexin43 During Electrical Uncoupling Induced by Ischemia
Authors: Beardslee Michael A. , Lerner Deborah L. , Tadros Peter N. , Laing James G. , Beyer Eric C. , Yamada Kathryn A. , Kléber André G. , Schuessler Richard B. , Saffitz Jeffrey E.
Published: June 2012

Abstract
—Electrical uncoupling at gap junctions during acute myocardial ischemia contributes to conduction abnormalities and reentrant arrhythmias. Increased levels of intracellular Ca
2+
and H
+
and accumulation of amphipathic lipid metabolites during ischemia promote uncoupling, but other mechanisms may play a role. We tested the hypothesis that uncoupling induced by acute ischemia is associated with changes in phosphorylation of the major cardiac gap junction protein, connexin43 (Cx43). Adult rat hearts perfused on a Langendorff apparatus were subjected to ischemia or ischemia/reperfusion. Changes in coupling were monitored by measuring whole-tissue resistance. Changes in the amount and distribution of phosphorylated and nonphosphorylated isoforms of Cx43 were measured by immunoblotting and confocal immunofluorescence microscopy using isoform-specific antibodies. In control hearts, virtually all Cx43 identified immunohistochemically at apparent intercellular junctions was phosphorylated. During ischemia, however, Cx43 underwent progressive dephosphorylation with a time course similar to that of electrical uncoupling. The total amount of Cx43 did not change, but progressive reduction in total Cx43 immunofluorescent signal and concomitant accumulation of nonphosphorylated Cx43 signal occurred at sites of intercellular junctions. Functional recovery during reperfusion was associated with increased levels of phosphorylated Cx43. These observations suggest that uncoupling induced by ischemia is associated with dephosphorylation of Cx43, accumulation of nonphosphorylated Cx43 within gap junctions, and translocation of Cx43 from gap junctions into intracellular pools.
Vascular Matrix Metalloproteinase-2–Dependent Cleavage of Calcitonin Gene-Related Peptide Promotes Vasoconstriction
Authors: Fernandez-Patron Carlos , Stewart Ken G. , Zhang Yunlong , Koivunen Erkki , Radomski Marek W. , Davidge Sandra T.
Published: June 2012

Abstract
—Matrix metalloproteinase (MMP)-2 has been historically associated with the process of vascular remodeling through the cleavage of extracellular matrix proteins. However, we recently found that MMP-2 also cleaves the endothelium-derived peptide big endothelin-1, ET-1[1–38] and yields the novel vasoconstrictor ET-1[1–32]. We therefore investigated the effects of MMP-2 inhibitors as potential vasodilators. MMP inhibition with ortho-phenanthroline (0.3 to 30 μmol/L) induced vasorelaxation of isolated rat mesenteric arteries (maximum of relaxation=74.5±27.6% at 30 μmol/L). However, phosphoramidon (0.3 to 30 μmol/L), which inhibits some metalloenzymes, but not MMP-2, did not dilate the arteries. Selective inhibition of endogenous MMP-2 with the novel tissue-permeable cyclic peptide CTTHWGFTLC (CTT, 10 μmol/L) also caused vasorelaxation (by 85±6%), whereas STTHWGFTLS (10 μmol/L), an inactive CTT analogue, did not dilate the arteries. Interestingly, the vasorelaxation that results from MMP-2 inhibition was endothelium-independent. Thus, we examined whether MMP-2 acted on peptides derived from the smooth muscle or the perivascular nerves. Recombinant human MMP-2 cleaved calcitonin gene-related peptide (CGRP) specifically at the Gly
14
-Leu
15
peptide bond and reduced the vasodilatory potency of CGRP by 20-fold. Inhibition of MMP-2 increased the amount of intact CGRP in arteries and enhanced vasorelaxation induced by anandamide, which stimulates CGRP release. Vasorelaxation in response to MMP-2 inhibition was abolished by CGRP[8–37], a selective CGRP receptor antagonist, and by capsaicin, which depletes arterial perivascular nerves of CGRP. We conclude that vascular MMP-2 cleaves endogenous CGRP and promotes vasoconstriction. These data suggest a novel mechanism of regulating the vasoactive and, possibly, the neurohormonal actions of CGRP and establish MMP-2 as a modulator of vascular function.
Cyclin A Downregulation and p21
cip1
Upregulation Correlate With GATA-6–Induced Growth Arrest in Glomerular Mesangial Cells
Authors: Nagata Daisuke , Suzuki Etsu , Nishimatsu Hiroaki , Yoshizumi Masao , Mano Toshiaki , Walsh Kenneth , Sata Masataka , Kakoki Masao , Goto Atsuo , Omata Masao , Hirata Yasunobu
Published: June 2012

Abstract
—The GATA-6 transcription factor is reported to be expressed in vascular myocytes. Because glomerular mesangial cells (GMCs) and vascular myocytes have similar properties, we examined whether GATA-6 was expressed in cultured GMCs and whether overexpression of GATA-6 induced cell cycle arrest in GMCs, using a recombinant adenovirus that expresses GATA-6 (Ad GATA-6). GATA-6 expression in GMCs was downregulated when quiescent GMCs were stimulated by serum to reenter the cell cycle. [
3
H]thymidine uptake was inhibited in GMCs infected with Ad GATA-6 in a dose- and time-dependent manner. The expression of cyclin A protein was decreased and that of the cyclin-dependent kinase inhibitor p21
cip1
was increased in GMCs infected with Ad GATA-6. Although the expression of p21
cip1
transcripts did not change remarkably, p21
cip1
protein was stabilized in GMCs infected with Ad GATA-6, suggesting a post-transcriptional regulation of p21
cip1
expression. Northern blot analysis showed that expression of the cyclin A transcript was decreased in Ad GATA-6–infected cells, whereas this decrease of cyclin A was not observed in GMCs derived from p21
cip1
null mice. Our results demonstrate that GATA-6 is endogenously expressed in GMCs and that overexpression of GATA-6 can induce cell cycle arrest. Our results also show that GATA-6–induced cell cycle arrest is associated with inhibition of cyclin A expression and p21
cip1
upregulation. Finally, our results indicate that the GATA-6–induced suppression of cyclin A expression depends on the presence of p21
cip1
.
Sarcoplasmic Reticulum Ca
2+
Release Causes Myocyte Depolarization
Authors: Schlotthauer Klaus , Bers Donald M.
Published: June 2012

Abstract
—Spontaneous sarcoplasmic reticulum (SR) Ca
2+
release causes delayed afterdepolarizations (DADs) via Ca
2+
-induced transient inward currents (
I
ti
). However, no quantitative data exists regarding (1) Ca
2+
dependence of DADs, (2) Ca
2+
required to depolarize the cell to threshold and trigger an action potential (AP), or (3) relative contributions of Ca
2+
-activated currents to DADs. To address these points, we evoked SR Ca
2+
release by rapid application of caffeine in indo 1-AM–loaded rabbit ventricular myocytes and measured caffeine-induced DADs (cDADs) with whole-cell current clamp. The SR Ca
2+
load of the myocyte was varied by different AP frequencies. The cDAD amplitude doubled for every 88±8 nmol/L of Δ[Ca
2+
]
i
(simple exponential), and the Δ[Ca
2+
]
i
threshold of 424±58 nmol/L was sufficient to trigger an AP. Blocking Na
+
-Ca
2+
exchange current (
I
Na/Ca
) by removal of [Na]
o
and [Ca
2+
]
o
(or with 5 mmol/L Ni
2+
) reduced cDADs by >90%, for the same Δ[Ca
2+
]
i
. In contrast, blockade of Ca
2+
-activated Cl
–
current (
I
Cl(Ca)
) with 50 μmol/L niflumate did not significantly alter cDADs. We conclude that DADs are almost entirely due to
I
Na/Ca
, not
I
Cl(Ca)
or Ca
2+
-activated nonselective cation current. To trigger an AP requires 30 to 40 μmol/L cytosolic Ca
2+
or a [Ca
2+
]
i
transient of 424 nmol/L. Current injection, simulating
I
ti
s with different time courses, revealed that faster
I
ti
s require less charge for AP triggering. Given that spontaneous SR Ca
2+
release occurs in waves, which are slower than cDADs or fast
I
ti
s, the true Δ[Ca
2+
]
i
threshold for AP activation may be ≈3-fold higher in normal myocytes. This provides a safety margin against arrhythmia in normal ventricular myocytes.
Cyclophilin A Is a Secreted Growth Factor Induced by Oxidative Stress
Authors: Jin Zheng-Gen , Melaragno Matthew G. , Liao Duan-Fang , Yan Chen , Haendeler Judith , Suh Young-Ah , Lambeth J. David , Berk Bradford C.
Published: June 2012

Abstract
—Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis, hypertension, and restenosis, in part by promoting vascular smooth muscle cell (VSMC) growth. Many VSMC growth factors are secreted by VSMC and act in an autocrine manner. Here we demonstrate that cyclophilin A (CyPA), a member of the immunophilin family, is secreted by VSMCs in response to oxidative stress and mediates extracellular signal–regulated kinase (ERK1/2) activation and VSMC growth by reactive oxygen species. Human recombinant CyPA can mimic the effects of secreted CyPA to stimulate ERK1/2 and cell growth. The peptidyl-prolyl isomerase activity is required for ERK1/2 activation by CyPA. In vivo, CyPA expression and secretion are increased by oxidative stress and vascular injury. These findings are the first to identify CyPA as a secreted redox-sensitive mediator, establish CyPA as a VSMC growth factor, and suggest an important role for CyPA and enzymes with peptidyl-prolyl isomerase activity in the pathogenesis of vascular diseases.
The Role of Electroporation in Defibrillation
Authors: Al-Khadra Ayman , Nikolski Vladimir , Efimov Igor R.
Published: June 2012

Abstract
—Electric shock is the only effective therapy against ventricular fibrillation. However, shocks are also known to cause electroporation of cell membranes. We sought to determine the impact of electroporation on ventricular conduction and defibrillation. We optically mapped electrical activity in coronary-perfused rabbit hearts during electric shocks (50 to 500 V). Electroporation was evident from transient depolarization, reduction of action potential amplitude, and upstroke dV/dt. Electroporation was voltage dependent and significantly more pronounced at the endocardium versus the epicardium, with thresholds of 229±81 versus 318±84 V, respectively (
P
=0.01, n=10), both being above the defibrillation threshold of 181.3±45.8 V. Epicardial electroporation was localized to a small area near the electrode, whereas endocardial electroporation was observed at the bundles and trabeculas throughout the entire endocardium. Higher-resolution imaging revealed that papillary muscles (n=10) were most affected. Electroporation and conduction block thresholds in papillary muscles were 281±64 V and 380±79 V, respectively. We observed no arrhythmia in association with electroporation. Further, preconditioning with high-energy shocks prevented reinduction of fibrillation by 50-V shocks, which were otherwise proarrhythmic. Endocardial bundles are the most susceptible to electroporation and the resulting conduction impairment. Electroporation is not associated with proarrhythmic effects and is associated with a reduction of vulnerability.
Embryonic Epinephrine Synthesis in the Rat Heart Before Innervation
Authors: Ebert Steven N. , Thompson Robert P.
Published: June 2012

Abstract
—Epinephrine is a potent neurotransmitter and hormone that can influence cardiac performance beginning shortly after the first myocardial contractions occur in developing vertebrate embryos. In the present study, we provide evidence that the heart itself may produce epinephrine during embryonic development. Using antibodies that selectively recognize the catecholamine biosynthetic enzymes, tyrosine hydroxylase, dopamine β-hydroxylase, and phenylethanolamine N-methyltransferase, we used coimmunofluorescent staining techniques to identify cardiac cells that have the capability of producing catecholamines. Initially, cells expressing catecholamine biosynthetic enzymes were found interspersed throughout the myocardium, but by embryonic day 11.5 (E11.5), they became preferentially localized to the dorsal venous valve and atrioventricular canal regions. As development proceeded, catecholamine biosynthetic enzyme expression decreased in these regions but became quite strong along the crest of the interventricular septum by E16.5. This expression pattern was also transient, decreasing in the ventricular septum by E19.5. These data are consistent with a transient and progressive association of catecholamine-producing cells within regions of the heart that become the sinoatrial node, atrioventricular node, and bundle of His. This is the first evidence demonstrating that intrinsic cardiac adrenergic cells may be preferentially associated with early pacemaking and conduction tissue development.
Interactions Between Nitric Oxide and Lipid Oxidation Pathways
Authors: O’Donnell Valerie B. , Freeman Bruce A.
Published: June 2012

Abstract
—Nitric oxide (
·
NO) signaling pathways and lipid oxidation reactions are of central importance in both the maintenance of vascular homeostasis and the progression of vascular disease. Because both of these pathways involve free radical species that can also react together at extremely fast rates, convergent interactions between these pathways are expected. Biochemical and cell biology studies have defined multiple interactions of
·
NO with oxidizing lipids that could lead to either vascular protection or potentiation of inflammatory vascular injury. For example, low levels of
·
NO generated by endothelial nitric oxide synthase can terminate propagating lipid radicals and inhibit lipoxygenases, reactions that would be protective. Alternatively, if generated at elevated levels, for example, after inducible nitric oxide synthase expression in inflammation,
·
NO can be converted to prooxidant species, such as peroxynitrite (ONOO
–
) and nitrogen dioxide (
·
NO
2
), that can potentiate inflammatory injury to vascular cells. Finally, both enzymatic and nonenzymatic lipid oxidation reactions can influence
·
NO bioactivity by directly scavenging
·
NO or altering the induction and catalytic activity of nitric oxide synthase enzymes. In this review, we summarize the biochemical interactions between
·
NO and lipid oxidation reactions and discuss the recognized and potential roles of these reactions in the vasculature.
Angiotensin AT
1
and AT
2
Receptors Differentially Regulate Angiopoietin-2 and Vascular Endothelial Growth Factor Expression and Angiogenesis by Modulating Heparin Binding–Epidermal Growth Factor (EGF)–Mediated EGF Receptor Transactivation
Authors: Fujiyama Soichiro , Matsubara Hiroaki , Nozawa Yoshihisa , Maruyama Katsuya , Mori Yasukiyo , Tsutsumi Yoshiaki , Masaki Hiroya , Uchiyama Yoko , Koyama Yoko , Nose Atsuko , Iba Osamu , Tateishi Eriko , Ogata Nahoko , Jyo Nobuo , Higashiyama Shigeki , Iwasaka Toshiji
Published: June 2012

Abstract
—Angiotensin II (Ang II)–mediated signals are transmitted via heparin binding epidermal growth factor (EGF)–like growth factor (HB-EGF) release followed by transactivation of EGF receptor (EGFR). Although Ang II and HB-EGF induce angiogenesis, their link to the angiopoietin (Ang)–Tie2 system remains undefined. We tested the effects of Ang II on Ang1, Ang2, or Tie2 expression in cardiac microvascular endothelial cells expressing the Ang II receptors AT
1
and AT
2
. Ang II significantly induced Ang2 mRNA accumulations without affecting Ang1 or Tie2 expression, which was inhibited by protein kinase C inhibitors and by intracellular Ca
2+
chelating agents. Ang II transactivated EGFR via AT
1
, and inhibition of EGFR abolished the induction of Ang2. Ang II caused processing of pro–HB-EGF in a metalloproteinase-dependent manner to stimulate maturation and release of HB-EGF. Neutralizing anti–HB-EGF antibody blocked EGFR phosphorylation by Ang II. Ang II also upregulated vascular endothelial growth factor (VEGF) expression in an HB-EGF/EGFR–dependent manner. AT
2
inhibited AT
1
-mediated Ang2 expression and phosphorylation of EGFR. In an in vivo corneal assay, AT
1
induced angiogenesis in an HB-EGF–dependent manner and enhanced the angiogenic activity of VEGF. Although neither Ang2 nor Ang1 alone induced angiogenesis, soluble Tie2-Fc that binds to angiopoietins attenuated AT
1
-mediated angiogenesis. These findings suggested that (1) Ang II induces Ang2 and VEGF expression without affecting Ang1 or Tie2 and (2) AT
1
stimulates processing of pro–HB-EGF by metalloproteinases, and the released HB-EGF transactivates EGFR to induce angiogenesis via the combined effect of Ang2 and VEGF, whereas AT
2
attenuates them by blocking EGFR phosphorylation. Thus, Ang II is involved in the VEGF-Ang-Tie2 system via HB-EGF–mediated EGFR transactivation, and this link should be considerable in pathological conditions in which collateral blood flow is required.
Functional Proteomic Analysis of Protein Kinase C ε Signaling Complexes in the Normal Heart and During Cardioprotection
Authors: Ping Peipei , Zhang Jun , Pierce William M. , Bolli Roberto
Published: June 2012

Abstract—
Using two-dimensional electrophoresis, mass spectrometry, immunoblotting, and affinity pull-down assays, we found that myocardial protein kinase C ε (PKCε) is physically associated with at least 36 known proteins that are organized into structural proteins, signaling molecules, and stress-responsive proteins. Furthermore, we found that the cardioprotection induced by activation of PKCε is coupled with dynamic modulation and recruitment of PKCε-associated proteins. The results suggest heretofore-unrecognized functions of PKCε and provide an integrated framework for the understanding of PKCε-dependent signaling architecture and cardioprotection.
Hyaluronan Enhances Contraction of Collagen by Smooth Muscle Cells and Adventitial Fibroblasts
Authors: Travis Jeffrey A. , Hughes Michael G. , Wong James M. , Wagner William D. , Geary Randolph L.
Published: June 2012

Abstract
—Remodeling contributes to restenosis when cells shrink the artery wall at sites of injury. This may be analogous to wound healing, where tissue remodeling achieves wound contraction. Hyaluronan (HA) is prominent in wound matrix and inhibits fetal scarring. HA is also produced in the artery wall after angioplasty, where it may inhibit constrictive remodeling. This hypothesis was tested in vitro using a model of matrix contraction. Primate aortic smooth muscle cells and adventitial fibroblasts were seeded into collagen I gels containing increasing amounts of HA (0% to 50%, wt/wt). Both cell types reduced the diameter of collagen alone ≈65% at 18 hours. HA significantly increased gel contraction (diameter in mm: 0% HA, 7.7±0.9; 2%, 7.1±0.7; 10%, 6.7±0.5; 50%, 5.6±0.9;
P
<0.05 for ≥10%), cell spreading and telopodia, and pericellular accumulation of collagen fibrils. These effects were mediated in part by cellular HA binding, because an antibody against CD44 receptors blocked pericellular collagen accumulation and enhanced gel contraction without altering cell shape. The role of CD44 was specific, because inhibiting receptor for hyaluronic acid–mediated motility (RHAMM) had no effect. Blocking β
1
-integrins completely inhibited contraction of collagen, but gels containing HA required CD44 and β
1
-integrin blockade for complete inhibition. Enhanced collagen reorganization and contraction were not attributable to increased collagenase activity, because the metalloproteinase inhibitor batimastat had no effect. In summary, HA enhanced collagen reorganization by the cell types most likely to mediate constrictive remodeling after angioplasty. These effects were CD44-dependent, thus providing a potential target for therapies to prevent constrictive remodeling and restenosis.
TrpC1 Is a Membrane-Spanning Subunit of Store-Operated Ca
2+
Channels in Native Vascular Smooth Muscle Cells
Authors: Xu Shang-Zhong , Beech David J.
Published: June 2012

Abstract
—Mammalian counterparts of the
Drosophila trp
gene have been suggested to encode store-operated Ca
2+
channels. These specialized channels are widely distributed and may have a general function to reload Ca
2+
into sarcoplasmic reticulum as well as specific functions, including the control of cell proliferation and muscle contraction. Heterologous expression of mammalian
trp
genes enhances or generates Ca
2+
channel activity, but the crucial question of whether any of the genes encode native subunits of store-operated channels remains unanswered. We have investigated if TrpC1 protein (encoded by
trp1
gene) is a store-operated channel in freshly isolated smooth muscle cells of resistance arterioles, arteries, and veins from human, mouse, or rabbit. Messenger RNA encoding TrpC1 was broadly expressed. TrpC1-specific antibody targeted to peptide predicted to contribute to the outer vestibule of TrpC1 channels revealed that TrpC1 is localized to the plasma membrane and has an extracellular domain. Peptide-specific binding of the antibody had a functional effect, selectively blocking store-operated Ca
2+
channel activity. The antibody is a powerful new tool for the study of mammalian
trp1
gene product. The study shows that TrpC1 is a novel physiological Ca
2+
channel subunit in arterial smooth muscle cells.
The Dual-Specificity Phosphatase MKP-1 Limits the Cardiac Hypertrophic Response In Vitro and In Vivo
Authors: Bueno Orlando F. , De Windt Leon J. , Lim Hae W. , Tymitz Kevin M. , Witt Sandra A. , Kimball Thomas R. , Molkentin Jeffery D.
Published: June 2012

Abstract
—Mitogen-activated protein kinase (MAPK) signaling pathways are important regulators of cell growth, proliferation, and stress responsiveness. A family of dual-specificity MAP kinase phosphatases (MKPs) act as critical counteracting factors that directly regulate the magnitude and duration of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. Here we show that constitutive expression of MKP-1 in cultured primary cardiomyocytes using adenovirus-mediated gene transfer blocked the activation of p38, JNK1/2, and ERK1/2 and prevented agonist-induced hypertrophy. Transgenic mice expressing physiological levels of MKP-1 in the heart showed (1) no activation of p38, JNK1/2, or ERK1/2; (2) diminished developmental myocardial growth; and (3) attenuated hypertrophy in response to aortic banding and catecholamine infusion. These results provide further evidence implicating MAPK signaling factors as obligate regulators of cardiac growth and hypertrophy and demonstrate the importance of dual-specificity phosphatases as counterbalancing regulatory factors in the heart.
Upregulation of the Nitric Oxide–cGMP Pathway in Aged Myocardium
Authors: Zieman Susan J. , Gerstenblith Gary , Lakatta Edward G. , Rosas Gisele O. , Vandegaer Koenraad , Ricker Kelly M. , Hare Joshua M.
Published: June 2012

Abstract
—Cardiovascular aging is associated with decreased endothelial vasoreactivity and prolonged diastolic relaxation. As diminished NO signaling contributes to age-associated endothelial dysfunction, we tested the hypothesis that impaired NO signaling or bioactivity also contributes to slowed ventricular relaxation with age. Accordingly, we measured myocardial NO synthase (NOS) enzyme activity, protein abundance, and cGMP production in old (22 to 25 months) and young adult (4 to 7 months) male Wistar rats. Both NOS3 protein abundance and calcium-dependent NOS activity were elevated in old compared with young adult hearts (7.2±1.1 versus 4.2±0.6 pmol/mg protein, respectively,
P
=0.03). However, NOS activity and protein abundance were similar in isolated myocytes, indicating that endothelial NOS likely explains the age difference. Cardiac effluent cGMP (enzyme immunoassay) was 4.8-fold higher (1794±373 fmol/min per mg heart tissue) in older versus younger hearts (
P
=0.003). To assess NO pathway responsiveness, we administered the NOS substrate l-arginine (100 μm) to isolated perfused rat hearts. Baseline isovolumic relaxation (τ) was prolonged in old (42.9±2.5 ms, n=16) versus young hearts (36.0±1.9 ms, n=11,
P
=0.03). l-Arginine decreased τ (
P
<0.001) and left ventricular end-diastolic pressure in both old and young hearts. Supporting an NO/cGMP-mediating mechanism, the NO donor sodium nitroprusside reduced τ (maximal effect, −14±2%, n=5,
P
<0.001), and this lusitropic effect was attenuated by the soluble guanylyl cyclase inhibitor 1
H
-[1,2,4]oxadiazolo-[4,3,-
a
]quinoxalin-1-one (n=7,
P
<0.001). Thus, the NO-cGMP pathway is upregulated in the endothelial cells of aged hearts. l-Arginine, the NOS precursor, enhances ventricular relaxation in old and young hearts, indicating that the NOS pathway may be exploited to modulate diastolic function in aged myocardium.
Vascular Trauma Induces Rapid but Transient Mobilization of VEGFR2
+
AC133
+
Endothelial Precursor Cells
Authors: Gill Muhammad , Dias Sergio , Hattori Koichi , Rivera Mary Lee , Hicklin Daniel , Witte Larry , Girardi Leonard , Yurt Roger , Himel Harvey , Rafii Shahin
Published: June 2012

Abstract
—Bone marrow (BM)–derived circulating endothelial precursor cells (CEPs) are thought to play a role in postnatal angiogenesis. Emerging evidence suggests that angiogenic stress of vascular trauma may induce mobilization of CEPs to the peripheral circulation. In this regard, we studied the kinetics of CEP mobilization in two groups of patients who experienced acute vascular insult secondary to burns or coronary artery bypass grafting (CABG). In both burn and CABG patients, there was a consistent, rapid increase in the number of CEPs, determined by their surface expression pattern of vascular endothelial growth factor receptor 2 (VEGFR2), vascular endothelial cadherin (VE-cadherin), and AC133. Within the first 6 to 12 hours after injury, the percentage of CEPs in the peripheral blood of burn or CABG patients increased almost 50-fold, returning to basal levels within 48 to 72 hours. Mobilized cells also formed late-outgrowth endothelial colonies (CFU-ECs) in culture, indicating that a small, but significant, number of circulating endothelial cells were BM-derived CEPs. In parallel to the mobilization of CEPs, there was also a rapid elevation of VEGF plasma levels. Maximum VEGF levels were detected within 6 to 12 hours of vascular trauma and decreased to baseline levels after 48 to 72 hours. Acute elevation of VEGF in the mice plasma resulted in a similar kinetics of mobilization of VEGFR2
+
cells. On the basis of these results, we propose that vascular trauma may induce release of chemokines, such as VEGF, that promotes rapid mobilization of CEPs to the peripheral circulation. Strategies to improve the mobilization and incorporation of CEPs may contribute to the acceleration of vascularization of the injured vascular tissue.
xIAP Induces Cell-Cycle Arrest and Activates Nuclear Factor-κB
Authors: Levkau Bodo , Garton Kyle J. , Ferri Nicola , Kloke Kerstin , Nofer Jerzy-Roch , Baba Hideo A. , Raines Elaine W. , Breithardt Günter
Published: June 2012

Abstract
—Survival of human vascular endothelial cells depends on their ability to activate the transcription factor nuclear factor-κB (NF-κB), a regulator of antiapoptotic genes, such as the X chromosome–linked inhibitor of apoptosis protein (xIAP). In the present study, we demonstrated expression of xIAP in the endothelial lining of normal human arteries and veins and elevated levels in highly malignant human endothelial tumors. Using retroviral infection of human endothelial cells, we identified two novel survival mechanisms mediated by xIAP in endothelial cells. First, xIAP can activate the transcription factor NF-κB, a known survival factor for human endothelial cells. This positive feedback loop induced by xIAP is mediated via phosphorylation and sustained degradation of inhibitor (I) κBα. Second, xIAP can inhibit cell proliferation via downregulation of cyclins A and D1 and induction of the cyclin-dependent kinase inhibitors p21
Cip1/Waf1
and p27
Kip1
. Cleavage of xIAP by caspases during endothelial cell apoptosis disables both of these biological functions of xIAP. Thus, caspase-mediated cleavage of xIAP interrupts a positive regulatory cytoprotective loop between NF-κB and xIAP and increases the vulnerability of the cell to apoptosis by releasing it from an xIAP-mediated quiescent state.
Thyroid Hormone Activation in Human Vascular Smooth Muscle Cells
Authors: Mizuma Haruo , Murakami Masami , Mori Masatomo
Published: June 2012

Abstract
—Thyroid hormone has been reported to have significant effects on the peripheral vascular system, including relaxation of vascular smooth muscle cells and antiatherosclerotic effects. To exert its biological activity, thyroxine, which is a major secretory product of thyroid gland, needs to be converted to 3,5,3′-triiodothyronine (T
3
) by iodothyronine deiodinase. Type I iodothyronine deiodinase (DI) is widely distributed and maintains circulating T
3
level, whereas type II iodothyronine deiodinase (DII) is present in a limited number of tissues to provide local intracellular T
3
. In the present study, we have identified iodothyronine deiodinase in cultured human coronary artery smooth muscle cells (hCASMCs) and human aortic smooth muscle cells (hASMCs). All of the characteristics of the deiodinating activity in hCASMCs and hASMCs were compatible with DII. Northern analysis demonstrated that DII mRNA was expressed in both hCASMCs and hASMCs, and DII mRNA levels as well as DII activities were rapidly increased by dibutyryl-cAMP or forskolin. These data demonstrate, for the first time, the expression of DII in human vascular smooth muscle cells, which is regulated by a cAMP-mediated mechanism. The present results suggest a previously unrecognized role of local T
3
production by DII in the pathophysiology of human vascular smooth muscle cells.
Chronic Nicotine Alters NO Signaling of Ca
2+
Channels in Cerebral Arterioles
Authors: Gerzanich Volodymyr , Zhang Fangyi , West G. Alexander , Simard J. Marc
Published: June 2012

Abstract
—Smoking is a major health hazard with proven deleterious effects on the cerebral circulation, including a decrease in cerebral blood flow and a high risk for stroke. To elucidate cellular mechanisms for the vasoconstrictive and pathological effects of nicotine, we used a nystatin-perforated patch-clamp technique to study Ca
2+
channels and Ca
2+
-activated K
+
(BK) channels in smooth muscle cells isolated from cerebral lenticulostriate arterioles of rats chronically exposed to nicotine (4.5 mg/kg per day of nicotine free base, 15 to 22 days via osmotic minipump). Two major effects were observed in cells from nicotine-treated animals compared with controls. First, Ca
2+
channels were upregulated (0.48±0.03 pS/pF [20 cells] versus 0.35±0.01 pS/pF [31 cells],
P
<0.005) and BK channels were downregulated (12±3 pA/pF [14 cells] versus 34±7 pA/pF [14 cells],
P
<0.05), mimicking the effect of an apparent decrease in bioavailability of endogenous NO. Second, normal downregulation of Ca
2+
channels by exogenous NO (sodium nitroprusside [SNP], 100 nmol/L) and cGMP (8-bromo-cGMP, 0.1 mmol/L) was absent, whereas normal upregulation of BK channels by these agents was preserved, suggesting block of NO signaling downstream of cGMP-dependent protein kinase. In pial window preparations, chronic nicotine blunted NO-induced vasodilation of pial vessels and the increase in cortical blood flow measured by laser-Doppler flowmetry, demonstrating the importance of Ca
2+
channel downregulation in NO-induced vasorelaxation. These findings elucidate a new pathophysiological mechanism involving altered Ca
2+
homeostasis in cerebral arterioles that may predispose to stroke.