Coronary Smooth Muscle Cells Research Articles

Characterization of circulating and monocyte-derived dendritic cells in obese and diabetic patients

Dendritic cells (DCs) are suspected to be involved in the development of atherogenesis, but their role is still unclear. The aim of this study was to characterize circulating DCs and monocyte-derived DCs (Mo-DCs) of obese and diabetic patients (T2D), and to study their interaction with human coronary smooth muscle cells (CASMCs). Obese post-menopausal women with or without insulin resistance were enrolled and were compared to age-matched healthy women. Myeloid circulating DCs significantly increased in obese T2D patients compared to healthy donors and a smaller increase was observed for plasmacytoid one. Mature Mo-DCs from obese T2D patients significantly decreased when compared to control, but they were significantly more capable of adhering to CASMCs compared to that from healthy controls and from not-T2D obese subjects. Altogether these data suggest that in conditions of insulin-resistance and obesity there is an up-regulation of myeloid DCs that might contribute to pathological vascular remodeling.

The identification of different endothelial cell populations within the mouse proepicardium

The proepicardium is a transient embryonic structure that is a source of precursors of the epicardium, coronary smooth muscle cells, and may be a source of coronary endothelial cells (EC). To better understand proepicardium development a systematic analysis of EC appearance was performed. Multiple marker analysis showed that EC are present in the mouse proepicardium at embryonic day (E) 9.0 through E9.75. Distinct populations of EC were found that were associated with the liver bud, and the sinus venosus, as well as a population that do not appear to be associated with either of these structures. There was a temporal increase in the number of EC and temporal changes in the distribution of EC within the different populations during PE development. These findings indicate that EC exist in the proepicardium before coronary vasculogenesis, and support a model in which there is a heterogeneous origin for EC in the proepicardium.

Low-intensity interval exercise training attenuates coronary vascular dysfunction and preserves Ca2+-sensitive K+ current in miniature swine with LV hypertrophy

Coronary vascular dysfunction has been observed in several models of heart failure (HF). Recent evidence indicates that exercise training is beneficial for patients with HF, but the precise intensity and underlying mechanisms are unknown. Left ventricular (LV) hypertrophy can play a significant role in the development of HF; therefore, the purpose of this study was to assess the effects of low-intensity interval exercise training on coronary vascular function in sedentary (HF) and exercise trained (HF-TR) aortic-banded miniature swine displaying LV hypertrophy. Six months postsurgery, in vivo coronary vascular responses to endothelin-1 (ET-1) and adenosine were measured in the left anterior descending coronary artery. Baseline and maximal coronary vascular conductance were similar between all groups. ET-1-induced reductions in coronary vascular conductance (P < 0.05) were greater in HF vs. sedentary control and HF-TR groups. Pretreatment with the ET type A (ET(A)) receptor blocker BQ-123 prevented ET-1 hypersensitivity in HF animals. Whole cell voltage clamp was used to characterize composite K(+) currents (I(K(+))) in coronary smooth muscle cells. Raising internal Ca(2+) from 200 to 500 nM increased Ca(2+)-sensitive K(+) current in HF-TR and control, but not HF animals. In conclusion, an ET(A)-receptor-mediated hypersensitivity to ET-1, elevated resting LV wall tension, and decreased coronary smooth muscle cell Ca(2+)-sensitive I(K(+)) was found in sedentary animals with LV hypertrophy. Low-intensity interval exercise training preserved normal coronary vascular function and smooth muscle cell Ca(2+)-sensitive I(K(+)), illustrating a potential mechanism underlying coronary vascular dysfunction in a large-animal model of LV hypertrophy. Our results demonstrate the potential clinical impact of exercise on coronary vascular function in HF patients displaying pathological LV hypertrophy.

Focal Adhesion Kinase Regulates Smooth Muscle Cell Recruitment to the Developing Vasculature

The investment of newly formed endothelial cell tubes with differentiated smooth muscle cells (SMC) is critical for appropriate vessel formation, but the underlying mechanisms remain unknown. We previously showed that depletion of focal adhesion kinase (FAK) in the nkx2.5 expression domain led to aberrant outflow tract (OFT) morphogenesis and strove herein to determine the cell types and mechanisms involved. We crossed fak(loxp) targeted mice with available Cre drivers to deplete FAK in OFT SMC (FAK(wnt) and FAK(nk)) or coronary SMC (FAK(cSMC)). In each case, depletion of FAK led to defective vasculogenesis that was incompatible with postnatal life. Immunohistochemical analysis of the mutant vascular structures revealed that FAK was not required for progenitor cell proliferation, survival, or differentiation into SMC but was necessary for subsequent SMC recruitment to developing vasculature. Using a novel FAK-null SMC culture model, we found that depletion of FAK did not influence SMC growth or survival, but blocked directional SMC motility and invasion toward the potent endothelial-derived chemokine, platelet-derived growth factor PDGFBB. FAK depletion resulted in unstable lamellipodial protrusions due to defective spatial-temporal activation of the small GTPase, Rac-1, and lack of Rac1-dependent recruitment of cortactin (an actin stabilizing protein) to the leading edge. Moreover, FAK null SMC exhibited a significant reduction in stimulated extracellular matrix degradation. FAK drives PDGFBB-stimulated SMC chemotaxis/invasion and is essential for SMC to appropriately populate the aorticopulmonary septum and the coronary vascular plexus.

Effects of rosuvastatin on the coordinated proteomic response of human coronary smooth muscle cells to low density lipoproteins

Introduction The clinical benefits of statins in reducing coronary events have been related to repopulation of the acellular core of atheromatous plaques with vascular smooth muscle cells (VSMC) and plaque stabilization. Intimal low-density lipoproteins (LDL) have been associated with VSMC phenotypic modulation and plaque destabilization. We have recently reported that LDL impair vascular remodelling because of changes affecting cytoskeleton proteins. Here, we used a proteomic approach to study whether rosuvastatin reverses the effects induced by atherogenic concentrations of LDL in human VSMC. Methods and Results VSMC treated with/without 10 μM rosuvastatin were incubated in the presence/absence of 100 μg/mL LDL for 24 h. Using 2DE and MALDI-ToF MS, we identified 39 non-redundant proteins (52 spots) with differential expression in LDL-treated cells. Thirteen of these proteins were reverted to normal levels by rosuvastatin. Thus, four proteins showed decreased intensity, six showed increased intensity, and three multispot proteins changed their distribution pattern in the rosuvastatin group. These proteins were identified as components of endoplasmic reticulum, mitochondrion, nucleus, cell membrane, cytoskeleton, and cytoplasm. In LDL-VSMC, rosuvastatin affected proteins involved in a variety of functions including cytoskeleton dynamics, chaperone activity, carbohydrate metabolism, protein biosynthesis and folding, kinase activity, redox processes, cytoprotective effects, and cell proliferation. Conclusions Our results demonstrate that the reversing effects of rosuvastatin in LDL-enriched VSMC affect functional proteins with a role in cellular metabolism and dynamics, which might contribute to the beneficial effects of statins by stabilizing lipid-rich atherosclerotic plaques in human atherosclerosis.

2′,3′-cAMP, 3′-AMP, and 2′-AMP inhibit human aortic and coronary vascular smooth muscle cell proliferation via A2B receptors

Rat vascular smooth muscle cells (VSMCs) from renal microvessels metabolize 2',3'-cAMP to 2'-AMP and 3'-AMP, and these AMPs are converted to adenosine that inhibits microvascular VSMC proliferation via A(2B) receptors. The goal of this study was to test whether this mechanism also exists in VSMCs from conduit arteries and whether it is similarly expressed in human vs. rat VSMCs. Incubation of rat and human aortic VSMCs with 2',3'-cAMP concentration-dependently increased levels of 2'-AMP and 3'-AMP in the medium, with a similar absolute increase in 2'-AMP vs. 3'-AMP. In contrast, in human coronary VSMCs, 2',3'-cAMP increased 2'-AMP levels yet had little effect on 3'-AMP levels. In all cell types, 2',3'-cAMP increased levels of adenosine, but not 5'-AMP, and 2',3'-AMP inhibited cell proliferation. Antagonism of A(2B) receptors (MRS-1754), but not A(1) (1,3-dipropyl-8-cyclopentylxanthine), A(2A) (SCH-58261), or A(3) (VUF-5574) receptors, attenuated the antiproliferative effects of 2',3'-cAMP. In all cell types, 2'-AMP, 3'-AMP, and 5'-AMP increased adenosine levels, and inhibition of ecto-5'-nucleotidase blocked this effect of 5'-AMP but not that of 2'-AMP nor 3'-AMP. Also, 2'-AMP, 3'-AMP, and 5'-AMP, like 2',3'-cAMP, exerted antiproliferative effects that were abolished by antagonism of A(2B) receptors with MRS-1754. In conclusion, VSMCs from conduit arteries metabolize 2',3'-cAMP to AMPs, which are metabolized to adenosine. In rat and human aortic VSMCs, both 2'-AMP and 3'-AMP are involved in this process, whereas, in human coronary VSMCs, 2',3'-cAMP is mainly converted to 2'-AMP. Because adenosine inhibits VSMC proliferation via A(2B) receptors, local vascular production of 2',3'-cAMP may protect conduit arteries from atherosclerosis.

In vitro epithelial-to-mesenchymal transformation in human adult epicardial cells is regulated by TGFβ-signaling and WT1

Adult epicardial cells are required for endogenous cardiac repair. After myocardial injury, they are reactivated, undergo epithelial-to-mesenchymal transformation (EMT) and migrate into the injured myocardium where they generate various cell types, including coronary smooth muscle cells and cardiac interstitial fibroblasts, which contribute to cardiac repair. To understand what drives epicardial EMT, we used an in vitro model for human adult epicardial cells. These cells have an epithelium-like morphology and markedly express the cell surface marker vascular cell adhesion marker (VCAM-1). In culture, epicardial cells spontaneously undergo EMT after which the spindle-shaped cells now express endoglin. Both epicardial cells before and after EMT express the epicardial marker, Wilms tumor 1 (WT1). Adding transforming growth factor beta (TGFβ) induces loss of epithelial character and initiates the onset of mesenchymal differentiation in human adult epicardial cells. In this study, we show that TGFβ-induced EMT is dependent on type-1 TGFβ receptor activity and can be inhibited by soluble VCAM-1. We also show that epicardial-specific knockdown of Wilms tumor-1 (WT1) induces the process of EMT in human adult epicardial cells, through transcriptional regulation of platelet-derived growth factor receptor alpha (Pdgfrα), Snai1 and VCAM-1. These data provide new insights into the process of EMT in human adult epicardial cells, which might provide opportunities to develop new strategies for endogenous cell-based cardiac repair.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-011-0181-0) contains supplementary material, which is available to authorized users.

Epicardial-Derived Cell Epithelial-to-Mesenchymal Transition and Fate Specification Require PDGF Receptor Signaling

In early heart development, platelet-derived growth factor (PDGF) receptor expression in the heart ventricles is restricted to the epicardium. Previously, we showed that PDGFRβ is required for coronary vascular smooth muscle cell (cVSMC) development, but a role for PDGFRα has not been identified. Therefore, we investigated the combined and independent roles of these receptors in epicardial development. To understand the contribution of PDGF receptors in epicardial development and epicardial-derived cell fate determination. By generating mice with epicardial-specific deletion of the PDGF receptors, we found that epicardial epithelial-to-mesenchymal transition (EMT) was defective. Sox9, an SRY-related transcription factor, was reduced in PDGF receptor-deficient epicardial cells, and overexpression of Sox9 restored epicardial migration, actin reorganization, and EMT gene expression profiles. The failure of epicardial EMT resulted in hearts that lacked epicardial-derived cardiac fibroblasts and cVSMC. Loss of PDGFRα resulted in a specific disruption of cardiac fibroblast development, whereas cVSMC development was unperturbed. Signaling through both PDGF receptors is necessary for epicardial EMT and formation of epicardial-mesenchymal derivatives. PDGF receptors also have independent functions in the development of specific epicardial-derived cell fates.

Monocyte chemotactic protein-3 induces human coronary smooth muscle cell proliferation

Monocyte chemotactic protein-3 (MCP-3), also known as CCL7, belongs to the monocyte chemotactic protein (MCP) subfamily of the CC chemokines that includes MCP-1/CCL2, MCP-2/CCL8, MCP-4/CCL13, and MCP-5/CCL12. Few studies have examined the role of MCP-3 in vascular pathologies such as atherosclerosis and restenosis in which smooth muscle cell (SMC) proliferation plays an important role. In this study, we investigated the effect of MCP-3 on human coronary artery smooth muscle cell (CASMC) proliferation. MCP-3 induced concentration-dependent CASMC proliferation with the maximum stimulatory effect at 0.3ng/mL (about 50% vs unstimulated cells) assessed by bromodeoxyuridine (BrdU) uptake and direct cell counting. Anti-MCP-3 antibody (20ng/mL) completely inhibited cell proliferation, demonstrating the specificity of the proliferative effect of MCP-3. Moreover, the MCP-3-induced CASMC proliferation was blocked by RS 102895 (0.06–6μM), a specific antagonist of chemokine receptor 2 (CCR2). The mitogenic effect of MCP-3 appeared to be dependent on ERK1/2 MAPK and PI3K signaling pathway activation, as demonstrated by the reduction of MCP-3-induced CASMC proliferation observed after the treatment of cells with U0126 (1μM) and LY-294002 (5μM), selective inhibitors of ERK 1/2 and PI3K activation, respectively. We found no relationship between MCP-3-induced CASMC proliferation and nuclear factor-κB activation. Moreover, we found that tumor necrosis factor-α (TNF-α, 30ng/mL) and interleukin-1β (IL-1β, 1ng/mL) both induced time-dependent increase of MCP-3 production by CASMCs, which was reduced by the anti-MCP-3 antibody (20ng/mL), suggesting that the mitogenic effect of these stimuli is due, at least in part, to MCP-3. In conclusion, our results demonstrate that MCP-3 is produced by human CASMCs and directly induces CASMC proliferation in vitro, suggesting a potential role for this chemokine in vascular pathology.

Mechanism related to docosahexaenoic acid induced large conductance calcium-activated potassium channel currents increase in coronary smooth muscle cells

To investigate the mechanism of enhanced large conductance calcium-activated potassium channel currents (BK) in coronary smooth muscle cells (SMCs) by docosahexaenoic acid (DHA). Coronary SMCs were isolated by enzyme digestion. Potassium channels in coronary SMCs were identified by applications of different potassium blockers. Effects of DHA and its metabolite 16, 17-epoxydocosapentaenoic acid (16, 17-EDP) on BK channels in the absence and presence of cytochrome P450 epoxygenase inhibitor SKF525A were studied by patch clamp in whole-cell configuration. BK channels were widely distributed in SMCs, and BK currents in normal SMCs accounted for (64.2 ± 2.7)% of total potassium currents (n = 20). DHA could activate BK channels, and its 50% effective concentration (EC(50)) was (0.23 ± 0.03) µmol/L, however, the effect of DHA on BK channels was abolished after SMCs were incubated with cytochrome P450 epoxygenase inhibitor SKF525A. 16, 17-EDP, a metabolite of DHA, could reproduce the effects of DHA on BK channels, and its EC(50) was (19.7 ± 2.8) nmol/L. DHA and metabolites can activate BK channels and dilate coronary arteries through activating cytochrome P450 epoxygenase pathway.

Expression of aromatase following angiotensin II stimulation in coronary vascular smooth muscle cells and cardiac fibroblasts

Aromatase converts testosterone to estrogen and is a key regulator of the balance between androgens and estrogens. Since sex steroids have been implicated in the outcome of cardiovascular disease, we investigated whether aromatase or androgen receptor (AR) expression was modified in human coronary vascular smooth muscle cells (HCVSM) and rat cardiac fibroblasts (RCF) following stimulation with a pathological mediator of heart disease (angiotensin II: AngII). Primary HCVSM (passage 8) and RCF (passage 0) were grown to 80% confluence and treated with AngII (24 hr; 10−7M). Cell type was characterized by expression of DDR2 (fibroblast) and α‐smooth muscle actin (HCVSM). We demonstrate that AngII increased expression of both aromatase (76%, p&lt;0.05) and androgen receptor (AR: 65%, p&lt;0.05) in HCVSM. In cardiac fibroblasts AngII produced a trend toward increased aromatase expression (23%, p=0.08) and although AR expression was not altered by AngII we are the first to report the presence of AR expression in cardiac fibroblasts. In conclusion, the presence of aromatase and AR in these two cell types suggests a possibility for circulating testosterone to act locally in an androgenic and/or estrogenic fashion. However the functional consequence of upregulation of both estrogenic (aromatase) and androgenic (AR) systems in HCVSM following AngII stimulation remains to be determined. Sarver Heart Center (TH) and AHA (RG).

Exercise attenuates reductions in intimal smooth muscle K + , but not Ca 2+ currents in a swine model of coronary artery disease

Exercise has been shown to reduce mortality and in some instances regress coronary lesions in humans with coronary artery disease (CAD). Smooth muscle cell (SMC) phenotypic modulation plays a central role in coronary lesion development, and we have shown that ion channel activity can alter coronary SMC phenotype. In the current study we hypothesized, that intimal SMCs (iSMC) would exhibit reduced K+ and Ca2+channel currents compared to non-diseased coronary SMC (cSMC), and that these reductions would be attenuated by exercise. For these experiments, Rapacz Familial Hypercholesterolemic (FH) swine underwent either a prevention (PEX) or treatment (TEX) exercise protocol. PEX swine were treadmill-trained for 4 months prior to being fed a high fat diet (n=7) for 6 months, whereas TEX swine were fed the high fat diet for 6 months prior to initiating exercise (n=8) for 4 months. Other FH swine were sedentary (SED; n=10) but also fed the high fat diet for 6 months. The weight matched control (con; n=5) group did not have CAD. FH swine had elevated cholesterol (778 ± 21 mg/dL), triglycerides (96.3 ± 3.4 mg/dL), and coronary lesions (43.1 ± 1.5 % plaque by IVUS). Reduced K+ current in iSMC of FH SED (19.5 ± 3.4 pA/pF, 100mV) compared to cSMC (84.3 ± 9.3), was attenuated by TEX (40.9 ± 7.9), but not PEX (20.1 ± 2.5). Conversely, reduced L-type Ca2+channel current in iSMC of FH SED (−0.8 ± 0.3 pA/pF, 20 mV), compared to cSMC (−4.9 ± 0.5), was not attenuated by TEX (−0.9 ± 0.1) nor PEX (−0.4 ± 0.2). In conclusion, exercise may attenuate SMC phenotypic modulation and lesion progression in patients with CAD by the maintaining functional K+ channels. NIH HL52490

KCa3.1 upregulated in familial hypercholesterolemic (FH) coronary smooth muscle

Familial hypercholesterolemia (FH) is a key risk factor for development of atherosclerosis. Vascular smooth muscle (VSMC) cell phenotypic modulation plays a key role in the development and stability of atherosclerotic plaques. We have previously shown upregulation of KCa3.1 channels mediates phenotypic modulation of coronary smooth muscle (CSM) cells and inhibition of these channels reduces restenosis following angioplasty. It remains unclear whether KCa3.1 channels are altered in atherosclerotic CSM of individuals with FH. Our objective was to determine whether KCa3.1 channels are upregulated in CSM isolated from FH swine. RCA sections from 2 year old FH swine showed a ~15 fold increase in artery stenosis, accompanied by elevated triglyceride and cholesterol values. In the media of FH coronaries, there was a trend for increased KCa3.1mRNA expression and KCa3.1 protein expression was elevated ~20%. KCa3.1 channel activity was also increased ~2‐fold in CSMCs isolated from FH swine. Our findings demonstrate that KCa3.1 is upregulated in CSM of FH swine and support previous research indicating KCa3.1 plays a key role in the development and progression of atherosclerosis.

Deletion of the Distal C Terminus of CaV1.2 Channels Leads to Loss of β-Adrenergic Regulation and Heart Failure in Vivo

L-type calcium currents conducted by CaV1.2 channels initiate excitation-contraction coupling in cardiac and vascular smooth muscle. In the heart, the distal portion of the C terminus (DCT) is proteolytically processed in vivo and serves as a noncovalently associated autoinhibitor of CaV1.2 channel activity. This autoinhibitory complex, with A-kinase anchoring protein-15 (AKAP15) bound to the DCT, is hypothesized to serve as the substrate for β-adrenergic regulation in the fight-or-flight response. Mice expressing CaV1.2 channels with the distal C terminus deleted (DCT-/-) develop cardiac hypertrophy and die prematurely after E15. Cardiac hypertrophy and survival rate were improved by drug treatments that reduce peripheral vascular resistance and hypertension, consistent with the hypothesis that CaV1.2 hyperactivity in vascular smooth muscle causes hypertension, hypertrophy, and premature death. However, in contrast to expectation, L-type Ca2+ currents in cardiac myocytes from DCT-/- mice were dramatically reduced due to decreased cell-surface expression of CaV1.2 protein, and the voltage dependence of activation and the kinetics of inactivation were altered. CaV1.2 channels in DCT-/- myocytes fail to respond to activation of adenylyl cyclase by forskolin, and the localized expression of AKAP15 is reduced. Therefore, we conclude that the DCT of CaV1.2 channels is required in vivo for normal vascular regulation, cell-surface expression of CaV1.2 channels in cardiac myocytes, and β-adrenergic stimulation of L-type Ca2+ currents in the heart.

H 2 O 2 dilates human coronary arterioles by stimulating the large‐conductance Ca 2+ ‐activated K + channel activity

H2O2 serves as an important endothelium-derived vasodilator factor in human coronary arterioles (HCAs) from patients with coronary artery disease (CAD). The cellular mechanisms by which H2O2 dilates HCAs remain unclear. Here we investigated the potential role of large-conductance Ca2+-activated K+ (BKCa) channels in the dilatory response to H2O2. Using RT-PCR and immunocytochemistry, the expression of BKCa was detected at both mRNA and protein levels in smooth muscle cells (SMCs) of HCAs. Western blot analysis of isolated arterioles revealed a higher level of BKCa (the pore-forming α subunit) protein expression in patients with CAD, as compared with those without CAD. Patch-clamp analysis identified a voltage- and paxilline-sensitive K+ current with a unitary conductance of 240-pS in freshly isolated coronary SMCs. Addition of H2O2 (50 μM) in the bath solution significantly activated this K+ current recorded from cell-attached patches (NPo, 0.089±0.043 vs. 0.0043±0.0037 of baseline; n=3), an effect that was subsequently blocked by paxilline (0.0035±0.0035; n=3). In isolated endothelium-denuded HCAs from CAD patients, H2O2 elicited a concentration-dependent dilation (dilation at 10−4 M, 98±2%; n=3). This dilation was markedly inhibited by paxilline (40±10%; n=3). In conclusion, the BKCa channel plays a key role in H2O2-induced dilation of HCAs in disease.

Inhibition of Transforming Growth Factor β Worsens Elastin Degradation in a Murine Model of Kawasaki Disease

Kawasaki disease (KD) is an acute inflammatory illness marked by coronary arteritis. However, the factors increasing susceptibility to coronary artery lesions are unknown. Because transforming growth factor (TGF) β increases elastin synthesis and suppresses proteolysis, we hypothesized that, in contrast to the benefit observed in aneurysms forming in those with Marfan syndrome, inhibition of TGF-β would worsen inflammatory-induced coronary artery lesions. By using a murine model of KD in which injection of Lactobacillus casei wall extract (LCWE) induces coronary arteritis, we show that LCWE increased TGF-β signaling in the coronary smooth muscle cells beginning at 2 days and continuing through 14 days, the point of peak coronary inflammation. By 42 days, LCWE caused fragmentation of the internal and external elastic lamina. Blocking TGF-β by administration of a neutralizing antibody accentuated the LCWE-mediated fragmentation of elastin and induced an overall loss of medial elastin without increasing the inflammatory response. We attributed these increased pathological characteristics to a reduction in the proteolytic inhibitor, plasminogen activator inhibitor-1, and an associated threefold increase in matrix metalloproteinase 9 activity compared with LCWE alone. Therefore, our data demonstrate that in the coronary arteritis associated with KD, TGF-β suppresses elastin degradation by inhibiting plasmin-mediated matrix metalloproteinase 9 activation. Thus, strategies to block TGF-β, used in those with Marfan syndrome, are unlikely to be beneficial and could be detrimental.

Notch Signaling Regulates Smooth Muscle Differentiation of Epicardium-Derived Cells

The embryonic epicardium plays a crucial role in the formation of the coronary vasculature and in myocardial development, yet the exact contribution of epicardium-derived cells (EPDCs) to the vascular and connective tissue of the heart, and the factors that regulate epicardial differentiation, are insufficiently understood. To define the role of Notch signaling in murine epicardial development. Using in situ hybridization and RT-PCR analyses, we detected expression of a number of Notch receptor and ligand genes in early epicardial development, as well as during formation of coronary arteries. Mice with epicardial deletion of Rbpj, the unique intracellular mediator of Notch signaling, survived to adulthood and exhibited enlarged coronary venous and arterial beds. Using a Tbx18-based genetic lineage tracing system, we show that EPDCs give rise to fibroblasts and coronary smooth muscle cells (SMCs) but not to endothelial cells in the wild type, whereas in Rbpj-deficient embryos EPDCs form and surround the developing arteries but fail to differentiate into SMCs. Conditional activation of Notch signaling results in premature SMC differentiation of epicardial cells and prevents coronary angiogenesis. We further show that Notch signaling regulates, and cooperates with transforming growth factor β signaling in SM differentiation of EPDCs. Notch signaling is a crucial regulator of SM differentiation of EPDCs, and thus, of formation of a functional coronary system.

Randomized, Double-Blind, Placebo-Controlled, Single Intravenous Dose-Escalation Study to Evaluate the Safety, Tolerability, and Pharmacokinetics of the Novel Coronary Smooth Muscle Cell Proliferation Inhibitor Biolimus A9 in Healthy Individuals

Biolimus A9 (BA9) is a novel proliferation inhibitor of coronary smooth muscle cells that has been specifically designed for coating drug-eluting stents. The goals of this study were to identify the highest safe intravenous dose of BA9, to evaluate the dose-dependent pharmacokinetics of BA9 after intravenous administration in humans, and to characterize early clinical symptoms of BA9 toxicity in healthy subjects. This phase 1 trial in healthy subjects was designed as a double-blind, placebo-controlled, randomized, ascending single-dose study. After screening and randomization, 28 volunteers received either placebo (n = 7) or BA9 (n = 21) in a double-blinded fashion. Doses from 0.0075 mg/kg were escalated to 0.25 mg/kg in 4 cohorts. BA9 concentrations were measured using liquid chromatography-tandem mass spectrometry. BA9 doses up to 0.075 mg/kg were well tolerated. Only the highest BA9 dose of 0.25 mg/kg produced reversible drug-related adverse events. The most frequent adverse events were headache, nausea, and mouth ulcers, most likely due to immunosuppression. Exposure to BA9 did not result in electrocardiographic or clinical laboratory changes. BA9 had a terminal half-life of 90.0 ± 40.0 hours (all n = 21, mean ± standard deviation), an apparent clearance from blood of 0.96 ± 1.07 L/kg/h, and a volume of distribution of 96.5 ± 72.6 L/kg.

Direct inhibitory effects of Ganciclovir on ICAM-1 expression and proliferation in human coronary vascular cells (SI/MPL-ratio: &gt;1)

SummaryBackgroundTreatment of the human cytomegalovirus (HCMV) infection with ganciclovir has beneficial indirect effects on the complex interactions of HCMV with restenosis, atherosclerosis, and transplant vascular sclerosis. The current study reports on direct effects of ganciclovir on expression of ICAM-1 and cell proliferation, key events of coronary atherosclerosis/restenosis. A potential clinical relevance of the data will be evaluated with the help of SI/MPL-ratio’s.Material/MethodsDefinition of the SI/MPL-ratio: relation between significant inhibitory effects in vitro/ex vivo and the maximal plasma level after systemic administration in vivo (ganciclovir: 9 μg/ml). Part I of the study investigated in cytoflow studies the effect of ganciclovir (0.05–5000 μg/mL) on TNF-a induced expression of intercellular adhesion molecule 1 (ICAM-1) in endothelial cells derived from umbilical veins (HUVEC), human coronary endothelial cells (HCAEC), and human coronary smooth muscle cells (HCMSMC). Part II of the study analysed the effect of ganciclovir (0.05–5000 μg/mL) on cell proliferation (HUVEC, HCAEC, and HCMSMC). In part III cytotoxic effects of ganciclovir (0.05–5000 μg/mL) were studied (HUVEC, HCAEC, and HCMSMC).ResultsGanciclovir caused slight but significant inhibitory effects on expression of ICAM-1 in HUVEC, HCAEC, and HCMSMC. In all three cell types studied strong dose depending significant antiproliferative effects of ganciclovir were detected. Partially, the antiproliferative effects of ganciclovir were caused by cytotoxic effects.ConclusionsSI/MPL-ratio’s >1 in HCAEC and HCMSMC indicate that the inhibitory effects of gancliclovir on ICAM-1-expression and cell proliferation may only be expected in vivo following local high dose administration e.g. in drug eluting stents (DES).

O-Glycosylation Regulates Ubiquitination and Degradation of the Anti-Inflammatory Protein A20 to Accelerate Atherosclerosis in Diabetic ApoE-Null Mice

BackgroundAccelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete.Methodology/Principal FindingsHigh glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis.ConclusionsHigh glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes.