Clinical relevance of endpoints in clinical trials for acid sphingomyelinase deficiency enzyme replacement therapy.

BACKGROUND: Acid sphingomyelinase deficiency (ASMD) also known as Niemann-Pick disease, is a rare lysosomal storage disorder with a diverse disease spectrum that includes slowly progressive, chronic visceral (type B) and neurovisceral forms (intermediate type A/B), in addition to infantile, rapidly progressive fatal neurovisceral disease (type A). PURPOSE AND METHODS: We review the published evidence on the relevance of splenomegaly and reduced lung diffusion capacity to the clinical burden of chronic forms of ASMD. Targeted literature searches were conducted to identify relevant ASMD and non-ASMD studies for associations between diffusing capacity of the lungs for carbon monoxide (DLCO) and splenomegaly, with clinical parameters and outcome measures. RESULTS: Respiratory disease and organomegaly are primary and independent contributors to mortality, disease burden, and morbidity for patients with chronic ASMD. The degree of splenomegaly correlates with short stature, atherogenic lipid profile, and degree of abnormality of hematologic parameters, and thus may be considered a surrogate marker for bleeding risk, abnormal lipid profiles and possibly, liver fibrosis. Progressive lung disease is a prevalent clinical feature of chronic ASMD, contributing to a decreased quality of life (QoL) and an increased disease burden. In addition, respiratory-related complications are a major cause of mortality in ASMD. CONCLUSIONS: The reviewed evidence from ASMD natural history and observational studies supports the use of lung function and spleen volume as clinically meaningful endpoints in ASMD trials that translate into important measures of disease burden for patients.

SDF-1 induces TNF-mediated apoptosis in cardiac myocytes.

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation. One such chemokine, Stromal cell-derived factor-1 (SDF-1) also known as CXCL12, and its receptor, CXCR4, are expressed and functional in cardiac myocytes. SDF-1 both stimulates and enhances the cellular signal which attracts potentially beneficial stem cells for tissue repair within the ischemic heart. Paradoxically however, this chemokine is known to act in concert with the inflammatory cytokines of the innate immune response which contributes to cellular injury through the recruitment of inflammatory cells during ischemia. In the present study, we have demonstrated that SDF-1 has dose dependent effects on freshly isolated cardiomyocytes. Using Tunnel and caspase 3-activation assays, we have demonstrated that the treatment of isolated adult rat cardiac myocyte with SDF-1 at higher concentrations (pathological concentrations) induced apoptosis. Furthermore, ELISA data demonstrated that the treatment of isolated adult rat cardiac myocyte with SDF-1 at higher concentrations upregulated TNF-α protein expression which directly correlated with subsequent apoptosis. There was a significant reduction in SDF-1 mediated apoptosis when TNF-α expression was neutralized which suggests that SDF-1 mediated apoptosis is TNF-α-dependent. The fact that certain stimuli are capable of driving cardiomyocytes into apoptosis indicates that these cells are susceptible to clinically relevant apoptotic triggers. Our findings suggest that the elevated SDF-1 levels seen in a variety of clinical conditions, including ischemic myocardial infarction, may either directly or indirectly contribute to cardiac cell death via a TNF-α mediated pathway. This highlights the importance of this receptor/ligand in regulating the cardiomyocyte response to stress conditions.

Na+/Ca2+ exchanger-1 protects against systolic failure in the Akitains2 model of diabetic cardiomyopathy via a CXCR4/NF-κB pathway.

Diabetic cardiomyopathy is characterized, in part, by calcium handling imbalances associated with ventricular dysfunction. The cardiac Na(+)/Ca(2+) exchanger 1 (NCX1) has been implicated as a compensatory mechanism in response to reduced contractility in the heart; however, its role in diabetic cardiomyopathy remains unknown. We aimed to fully characterize the Akita(ins2) murine model of type 1 diabetes through assessing cardiac function and NCX1 regulation. The CXCL12/CXCR4 chemokine axis is well described in its cardioprotective effects via progenitor cell recruitment postacute myocardial infarction; however, it also functions in regulating calcium dependent processes in the cardiac myocyte. We therefore investigated the potential impact of CXCR4 in diabetic cardiomyopathy. Cardiac performance in the Akita(ins2) mouse was monitored using echocardiography and in vivo pressure-volume analysis. The Akita(ins2) mouse is protected against ventricular systolic failure evident at both 5 and 12 mo of age. However, the preserved contractility was associated with a decreased sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a)/phospholamban ratio and increased NCX1 content. Direct myocardial injection of adenovirus encoding anti-sense NCX1 significantly decreased NCX1 expression and induced systolic failure in the Akita(ins2) mouse. CXCL12 and CXCR4 were both upregulated in the Akita(ins2) heart, along with an increase in IκB-α and NF-κB p65 phosphorylation. We demonstrated that CXCR4 activation upregulates NCX1 expression through a NF-κB-dependent signaling pathway in the cardiac myocyte. In conclusion, the Akita(ins2) type 1 diabetic model is protected against systolic failure due to increased NCX1 expression. In addition, our studies reveal a novel role of CXCR4 in the diabetic heart by regulating NCX1 expression via a NF-κB-dependent mechanism.

Effects of CXCR4 gene transfer on cardiac function after ischemia-reperfusion injury.

Acute coronary occlusion is the leading cause of death in the Western world. There is an unmet need for the development of treatments to limit the extent of myocardial infarction (MI) during the acute phase of occlusion. Recently, investigators have focused on the use of a chemokine, CXCL12, the only identified ligand for CXCR4, as a new therapeutic modality to recruit stem cells to individuals suffering from MI. Here, we examined the effects of overexpression of CXCR4 by gene transfer on MI. Adenoviruses carrying the CXCR4 gene were injected into the rat heart one week before ligation of the left anterior descending coronary artery followed by 24 hours reperfusion. Cardiac function was assessed by echocardiography couple with 2,3,5-Triphenyltetrazolium chloride staining to measure MI size. In comparison with control groups, rats receiving Ad-CXCR4 displayed an increase in infarct area (13.5% +/- 4.1%) and decreased fractional shortening (38% +/- 5%). Histological analysis revealed a significant increase in CXCL12 and tumor necrosis factor-alpha expression in ischemic area of CXCR4 overexpressed hearts. CXCR4 overexpression was associated with increased influx of inflammatory cells and enhanced cardiomyocyte apoptosis in the infarcted heart. These data suggest that in our model overexpressing CXCR4 appears to enhance ischemia/reperfusion injury possibly due to enhanced recruitment of inflammatory cells, increased tumor necrosis factor-alpha production, and activation of cell death/apoptotic pathways.

Hepatic stellate cells express functional CXCR4: role in stromal cell-derived factor-1alpha-mediated stellate cell activation.

UNLABELLED: Chemokine interactions with their receptors have been implicated in hepatic stellate cell (HSC) activation. The hepatic expression of CXCR4 messenger RNA is increased in hepatitis C cirrhotic livers and plasma levels of its endogenous ligand, stromal cell-derived factor-1alpha (SDF-1alpha), correlate with increased fibrosis in these patients. The expression of CXCR4 by HSCs has not been reported. We therefore examined whether HSCs express CXCR4 in vivo and in vitro and explored whether SDF-1alpha/CXCR4 receptor engagement promotes HSC activation, fibrogenesis, and proliferation. The hepatic protein expression of both CXCR4 and SDF-1alpha is increased in hepatitis C cirrhotic livers and immunoflourescent and immunohistochemical staining confirms that HSCs express CXCR4 in vivo. Immortalized human stellate cells as well as primary human HSCs express CXCR4, and cell surface receptor expression increases with progressive culture-induced activation. Treatment of stellate cells with recombinant SDF-1alpha increases expression of alpha-smooth muscle actin and collagen I and stimulates a dose-dependent increase in HSC proliferation. Inhibitor studies suggest that SDF-1alpha/CXCR4-dependent extracellular signal-regulated kinase 1/2 and Akt phosphorylation mediate effects on collagen I expression and stellate cell proliferation. CONCLUSION: HSCs express CXCR4 receptor in vivo and in vitro. CXCR4 receptor activation by SDF-1alpha is profibrogenic through its effects on HSC activation, fibrogenesis, and proliferation. Extracellular signal-regulated kinase 1/2 and phosphoinositide 3-kinase pathways mediate SDF-1alpha-induced effects on HSC expression of collagen I and proliferation. The availability of small molecule inhibitors of CXCR4 make this receptor an appealing target for antifibrotic approaches.

ß2-Adrenergic receptor signaling in the cardiac myocyte is modulated by interactions with CXCR4.

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation, metastasis, and embryonic development. We previously demonstrated a nonchemotactic role for one such chemokine pair, stromal cell-derived factor-1α and its G-protein coupled receptor, CXCR4. Stromal cell-derived factor-1/CXCR4 are expressed on cardiac myocytes and have direct consequences on cardiac myocyte physiology by inhibiting contractility in response to the nonselective ß-adrenergic receptor (ßAR) agonist, isoproterenol. As a result of the importance of ß-adrenergic signaling in heart failure pathophysiology, we investigated the underlying mechanism involved in CXCR4 modulation of ßAR signaling. Our studies demonstrate activation of CXCR4 by stromal cell-derived factor-1 leads to a decrease in ßAR-induced PKA activity as assessed by cAMP accumulation and PKA-dependent phosphorylation of phospholamban, an inhibitor of SERCA2a. We determined CXCR4 regulation of ßAR downstream targets is ß2AR-dependent. We demonstrated a physical interaction between CXCR4 and ß2AR as determined by coimmunoprecipitation, confocal microscopy, and BRET techniques. The CXCR4-ß2AR interaction leads to G-protein signal modulation and suggests the interaction is a novel mechanism for regulating cardiac myocyte contractility. Chemokines are physiologically and developmentally relevant to myocardial biology and represent a novel receptor class of cardiac modulators. The CXCR4-ß2AR complex could represent a hitherto unknown target for therapeutic intervention.

Quantitation of CXCR4 expression in myocardial infarction using 99mTc-labeled SDF-1alpha.

UNLABELLED: The chemokine stromal-derived factor-1alpha (SDF-1alpha, CXCL12) and its receptor CXCR4 are implicated as key mediators of hematopoietic stem cell retention, cancer metastasis, and HIV infection. Their role in myocardial infarction (MI) is not as well defined. The noninvasive in vivo quantitation of CXCR4 expression is central to understanding its importance in these diverse processes as well in the cardiac response to injury. METHODS: Recombinant SDF-1alpha was radiolabeled under aprotic conditions and purified by gel-filtration chromatography (GFC) using high-specific-activity 99mTc-S-acetylmercaptoacetyltriserine-N-hydroxysuccinimide ([99mTc-MAS3]-NHS) prepared by solid-phase preloading. Radiotracer stability and transmetallation under harsh conditions were quantified by GFC. Affinity, specificity, and maximum number of binding sites (Bmax) were quantified, with adenoviral-expressed CXCR4 on nonexpressing cells and endogenous receptor on rat neonatal cardiomyocytes, using a high-throughput live-cell-binding assay. Blood half-life, biodistribution, and clearance of intravenously injected [99mTc-MAS3]-SDF-1alpha were quantified in Sprague-Dawley rats before and after experimentally induced MI. RESULTS: [99mTc-MAS3]-SDF-1alpha could be prepared in 2 h total with a specific activity of 8.0 x 10(7) MBq/mmol (2,166 Ci/mmol) and a radiochemical purity greater than 98%. Degradation of the radiotracer after boiling for 5 min, with and without 1 mM dithiothreitol, and transmetallation in 100% serum at 37 degrees C for 4 h were negligible. [99mTc-MAS3]-SDF-1alpha exhibits high specificity for CXCR4 on the surface of living rat neonatal cardiomyocytes, with an affinity of 2.7 +/- 0.9 nM and a Bmax of 4.8 x 10(4) binding sites per cell. After intravenous injection, 99mTc-labeled SDF-1alpha displays a blood half-life of 25.8 +/- 4.6 min, rapid renal clearance with only 26.2 +/- 6.1 percentage injected dose remaining in the carcass at 2 h, consistently low uptake in most organs (<0.1 percentage injected dose per gram), and no evidence of blood-brain barrier penetration. After MI was induced, CXCR4 expression levels in the myocardium increased more than 5-fold, as quantified using [99mTc-MAS3]-SDF-1alpha and confirmed using confocal immunofluorescence. CONCLUSION: We describe a 99mTc-labeled SDF-1alpha radiotracer that can be used as a sensitive and specific probe for CXCR4 expression in vivo and demonstrate that this radiotracer is able to quantify changes in CXCR4 expression under different physiologic and pathologic states. Taken together, CXCR4 levels should now be quantifiable in vivo in a variety of animal model systems of human diseases.

Ambient air particulate matter exposure and tissue factor expression in atherosclerosis.

Recent studies have suggested a link between inhaled particulate matter (PM) exposure and atherogenesis. We investigated tissue factor (TF) expression with ambient fine particulate matter (diameter < 2.5 microm, PM(2.5)) exposure and in response to in vitro exposure to fine and ultrafine PM in cultured human bronchial epithelial cells, vascular smooth muscle cells (hSMCs), and monocytes. ApoE-/- mice, fed with normal chow (NC) or high-fat chow (HFC), were exposed to concentrated PM(2.5) or filtered air (FA) for 6 mo (6 h/day, 5 day/wk, n = 28). Following in vivo ultrasound bio-microscopy (UBM) assessment of plaque area, macrophage infiltration (CD68) and TF expression in the aorta were quantified. Cultured cells were incubated with size-fractionated PM from cascade impactors, or with standard reference PM material (SRM, number 1649a) and assayed for TF protein, mRNA, and activity. UBM-derived plaque areas were 7 +/- 1% larger in the PM(2.5)-HFC than the FA-HFC group (p = .04), but not significantly different between the PM(2.5)-NC and FA-NC groups (p = .07). Immunohistochemistry revealed increased TF (15 +/- 3% vs. 8 +/- 2%, p < .01) and macrophage infiltration (19 +/- 2% vs. 14 +/- 3%, p < .01) in the plaques of PM(2.5)-HFC compared with FA-HFC groups. Impactor-collected PM(2.5) and ultrafine particles consistently increased TF protein in bronchial epithelial cells, monocytes, and hSMCs. TF mRNA expression increased rapidly (within 1 h) in response to SRM PM. We conclude that in vivo and in vitro exposure to ambient air PM(2.5) induces TF expression.

Chemokines induce matrix metalloproteinase-2 through activation of epidermal growth factor receptor in arterial smooth muscle cells.

OBJECTIVE: Matrix metalloproteinases (MMP) are critical to smooth muscle cell (SMC) migration in vivo. MMP-2 dysregulation has been implicated in the pathogenesis of abnormal arterial remodeling, aneurysm formation, and atherosclerotic plaque structure and stability. The chemokine receptors CCR3 and CXCR4 are present and functional on SMC and are up-regulated in vascular diseases such as atherosclerosis. We sought to determine a potential mechanism for chemokine receptor-mediated effects on the vasculature by asking whether the chemokines eotaxin (CCL11), the ligand for CCR3, and stromal cell-derived cell factor (SDF-1, CXCL12), the ligand for CXCR4, induce MMP-2 in SMC. Studies were then performed to define the signaling pathways involved. METHODS AND RESULTS: As determined by RT-PCR, Western blotting and zymography, SDF-1 and eotaxin induce MMP-2 mRNA, protein, and activity in SMC. An anti-CCR3 antibody and a CXCR4 antagonist blocked proMMP-2 induction by SDF-1 and eotaxin, the respective ligands for the chemokine receptors CXCR4 and CCR3, suggesting that the inductions by these chemokines are receptor-mediated. Receptor cross-talk between G-protein-coupled receptors (GPCR) and the epidermal growth factor receptor (EGFR) is a method of expanding the GPCRs' signaling repertoire. We demonstrate, for the first time to our knowledge, that in SMC, chemokine induction of proMMP-2 is dependent on activation of the EGFR. Interestingly, by blocking the ligand binding domain of EGFR, we demonstrate that activation of EGFR by SDF-1 and eotaxin occurs through different cellular pathways. CONCLUSION: The pro-inflammatory chemokines eotaxin and SDF induce proMMP-2 activation of EGFR through two different pathways. SDF and eotaxin, as regulators of proMMP-2 expression and by engaging in receptor cross-talk, may play critical roles in atherosclerosis, restenosis, and plaque rupture. These ligands and their respective receptors, CXCR4 and CCR3, therefore may serve as future potential therapeutic targets.

Arterial Effects of Canakinumab in Patients With Atherosclerosis and Type 2 Diabetes or Glucose Intolerance.

BACKGROUND: Evidence suggests that interleukin (IL)-1ß is important in the pathogenesis of atherosclerosis and its complications and that inhibiting IL-1ß may favorably affect vascular disease progression. OBJECTIVES: The goal of this study was to evaluate the effects of IL-1ß inhibition with canakinumab versus placebo on arterial structure and function, determined by magnetic resonance imaging. METHODS: Patients (N = 189) with atherosclerotic disease and either type 2 diabetes mellitus or impaired glucose tolerance were randomized to receive placebo (n = 94) or canakinumab 150 mg monthly (n = 95) for 12 months. They underwent magnetic resonance imaging of the carotid arteries and aorta. RESULTS: There were no statistically significant differences between canakinumab compared with placebo in the primary efficacy and safety endpoints. There was no statistically significant change in mean carotid wall area and no effect on aortic distensibility, measured at 3 separate anatomic sites. The change in mean carotid artery wall area was -3.37 mm2 after 12 months with canakinumab versus placebo. High-sensitivity C-reactive protein was significantly reduced by canakinumab compared with placebo at 3 months (geometric mean ratio [GMR]: 0.568; 95% confidence interval [CI]: 0.436 to 0.740; p < 0.0001) and 12 months (GMR: 0.56; 95% CI: 0.414 to 0.758; p = 0.0002). Lipoprotein(a) levels were reduced by canakinumab compared with placebo (-4.30 mg/dl [range: -8.5 to -0.55 mg/dl]; p = 0.025] at 12 months), but triglyceride levels increased (GMR: 1.20; 95% CI: 1.046 to 1.380; p = 0.01). In these patients with type 2 diabetes mellitus or impaired glucose tolerance, canakinumab had no effect compared with placebo on any of the measures assessed by using a standard oral glucose tolerance test. CONCLUSIONS: There were no statistically significant effects of canakinumab on measures of vascular structure or function. Canakinumab reduced markers of inflammation (high-sensitivity C-reactive protein and interleukin-6), and there were modest increases in levels of total cholesterol and triglycerides. (Safety & Effectiveness on Vascular Structure and Function of ACZ885 in Atherosclerosis and Either T2DM or IGT Patients; NCT00995930).

CCL11 (Eotaxin) induces CCR3-dependent smooth muscle cell migration.

OBJECTIVE: CCL11 (Eotaxin) is a potent eosinophil chemoattractant that is abundant in atheromatous plaques. The major receptor for CCL11 is CCR3, which is found on leukocytes and on some nonleukocytic cells. We sought to determine whether vascular smooth muscle cells (SMCs) possessed functional CCR3. METHODS AND RESULTS: CCR3 mRNA (by RT-PCR) and protein (by Western blot analysis and flow cytometry) were present in mouse aortic SMCs. CCL11 induced concentration-dependent SMC chemotaxis in a modified Boyden chamber, with maximum effect seen at 100 ng/mL. SMC migration was markedly inhibited by antibody to CCR3, but not to CCR2. CCL11 also induced CCR3-dependent SMC migration in a scrape-wound assay. CCL11 had no effect on SMC proliferation. CCR3 and CCL11 staining were minimal in the normal arterial wall, but were abundant in medial SMC and intimal SMC 5 days and 28 days after mouse femoral arterial injury, respectively, times at which SMCs possess a more migratory phenotype. CONCLUSIONS: These data demonstrate that SMCs possess CCR3 under conditions associated with migration and that CCL11 is a potent chemotactic factor for SMCs. Because CCL11 is expressed abundantly in SMC-rich areas of the atherosclerotic plaque and in injured arteries, it may play an important role in regulating SMC migration.

MCP-1-dependent signaling in CCR2(-/-) aortic smooth muscle cells.

Monocyte chemoattractant protein-1 (MCP-1, CCL2) is a mediator of inflammation that has been implicated in the pathogenesis of a wide variety of human diseases. CCR2, a heterotrimeric G-coupled receptor, is the only known receptor that functions at physiologic concentrations of MCP-1. Despite the importance of CCR2 in mediating MCP-1 responses, several recent studies have suggested that there may be another functional MCP-1 receptor. Using arterial smooth muscle cells (SMC) from CCR2(-/-) mice, we demonstrate that MCP-1 induces tissue-factor activity at physiologic concentrations. The induction of tissue factor by MCP-1 is blocked by pertussis toxin and 1,2-bis(O-aminophenyl-ethane-ethan)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, suggesting that signal transduction through the alternative receptor is G(alphai)-coupled and dependent on mobilization of intracellular Ca(2+). MCP-1 induces a time- and concentration-dependent phosphorylation of the mitogen-activated protein kinases p42/44. The induction of tissue factor activity by MCP-1 is blocked by PD98059, an inhibitor of p42/44 activation, but not by SB203580, a selective p38 inhibitor. These data establish that SMC possess an alternative MCP-1 receptor that signals at concentrations of MCP-1 that are similar to those that activate CCR2. This alternative receptor may be important in mediating some of the effects of MCP-1 in atherosclerotic arteries and in other inflammatory processes.

Elevated osteoprotegerin is associated with abnormal ankle brachial indices in patients infected with HIV: a cross-sectional study.

BACKGROUND: Patients infected with HIV have an increased risk for accelerated atherosclerosis. Elevated levels of osteoprotegerin, an inflammatory cytokine receptor, have been associated with a high incidence of cardiovascular disease (including peripheral arterial disease, or PAD), acute coronary syndrome, and cardiovascular mortality. The objective of this study was to determine whether PAD is prevalent in an HIV-infected population, and to identify an association with HIV-specific and traditional cardiovascular risk factors, as well as levels of osteoprotegerin. METHODS: One hundred and two patients infected with HIV were recruited in a cross-sectional study. To identify the prevalence of PAD, ankle-brachial indices (ABIs) were measured. Four standard ABI categories were utilized: < or = 0.90 (definite PAD); 0.91-0.99 (borderline); 1.00-1.30 (normal); and >1.30 (high). Medical history and laboratory measurements were obtained to determine possible risk factors associated with PAD in HIV-infected patients. RESULTS: The prevalence of PAD (ABI < or = 0.90) in a young HIV-infected population (mean age: 48 years) was 11%. Traditional cardiovascular risk factors, including advanced age and previous cardiovascular history, as well as elevated C-reactive protein levels, were associated with PAD. Compared with patients with normal ABIs, patients with high ABIs had significantly elevated levels of osteoprotegerin [1428.9 (713.1) pg/ml vs. 3088.6 (3565.9) pg/ml, respectively, p = 0.03]. CONCLUSIONS: There is a high prevalence of PAD in young HIV-infected patients. A number of traditional cardiovascular risk factors and increased osteoprotegerin concentrations are associated with abnormal ABIs. Thus, early screening and aggressive medical management for PAD may be warranted in HIV-infected patients.

Association of HIV viral load with monocyte chemoattractant protein-1 and atherosclerosis burden measured by magnetic resonance imaging.

BACKGROUND: HIV-infected individuals may be at increased risk for atherosclerosis. Although this is partially attributable to metabolic factors, HIV-associated inflammation may play a role. OBJECTIVE: To investigate associations of HIV disease with serum monocyte chemoattractant protein-1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2) levels and atherosclerosis burden. DESIGN: A cross-sectional analysis. METHODS: : Serum MCP-1/CCL2, fasting lipids, and glucose tolerance were measured in 98 HIV-infected and 79 demographically similar uninfected adults. Eighty-four participants had MRI of the carotid arteries and thoracic aorta to measure atherosclerosis burden. Multivariate analyses were performed using linear regression. RESULTS: Mean MCP-1/CCL2 levels did not differ between HIV-infected and uninfected participants (P = 0.65). Among HIV-infected participants, after adjusting for age, BMI, and cigarette smoking, HIV-1 viral load was positively associated with MCP-1/CCL2 (P = 0.02). Multivariate analyses adjusting for sex, low-density lipoprotein cholesterol, total cholesterol:high-density lipoprotein cholesterol ratio, cigarette smoking, MCP-1/CCL2, and protease inhibitor use found that HIV infection was associated with greater mean thoracic aorta vessel wall area (VWA, P < 0.01) and vessel wall thickness (VWT, P = 0.03), but not with carotid artery parameters. Compared with being uninfected, having detectable HIV-1 viremia was associated with greater mean thoracic aorta VWA (P < 0.01) and VWT (P = 0.03), whereas being HIV-infected with undetectable viral load was associated with greater thoracic aorta VWA (P = 0.02) but not VWT (P = 0.15). There was an independent positive association of MCP-1/CCL2 with thoracic aorta VWA (P = 0.01) and VWT (P = 0.01). CONCLUSION: HIV-1 viral burden is associated with higher serum levels of MCP-1/CCL2 and with atherosclerosis burden, as assessed by thoracic aorta VWA and VWT.

Human immunodeficiency virus (HIV) infects human arterial smooth muscle cells in vivo and in vitro: implications for the pathogenesis of HIV-mediated vascular disease.

Human immunodeficiency virus (HIV) infection is associated with accelerated atherosclerosis and vasculopathy, although the mechanisms underlying these findings have not been determined. Hypotheses for these observations include: 1) an increase in the prevalence of established cardiac risk factors observed in HIV-infected individuals who are currently experiencing longer life expectancies; 2) the dyslipidemia reported with certain HIV anti-retroviral therapies; and/or 3) the proinflammatory effects of infiltrating HIV-infected monocytes/macrophages. An unexplored possibility is whether HIV itself can infect vascular smooth muscle cells (SMCs) and, by doing so, whether SMCs can accelerate vascular disease. Our studies demonstrate that human SMCs can be infected with HIV both in vivo and in vitro. The HIV protein p24 was detected by fluorescence confocal microscopy in SMCs from tissue sections of human atherosclerotic plaques obtained from HIV-infected individuals. Human SMCs could also be infected in vitro with HIV by a mechanism dependent on CD4, the chemokine receptors CXCR4 or CCR5, and endocytosis, resulting in a marked increase in SMC secretion of the chemokine CCL2/MCP-1, which has been previously shown to be a critical mediator of atherosclerosis. In addition, SMC proliferation appeared concentric to the vessel lumen, and minimal inflammation was detected, unlike typical atherosclerosis. Our data suggest that direct infection of human arterial SMCs by HIV represents a potential mechanism in a multifactorial paradigm to explain the exacerbated atherosclerosis and vasculopathy reported in individuals infected with HIV.

beta-Adrenergic receptor activation induces internalization of cardiac Cav1.2 channel complexes through a beta-arrestin 1-mediated pathway.

Voltage-dependent calcium channels (VDCCs) play a pivotal role in normal excitation-contraction coupling in cardiac myocytes. These channels can be modulated through activation of beta-adrenergic receptors (beta-ARs), which leads to an increase in calcium current (I(Ca-L)) density through cardiac Ca(v)1 channels as a result of phosphorylation by cAMP-dependent protein kinase A. Changes in I(Ca-L) density and kinetics in heart failure often occur in the absence of changes in Ca(v)1 channel expression, arguing for the importance of post-translational modification of these channels in heart disease. The precise molecular mechanisms that govern the regulation of VDCCs and their cell surface localization remain unknown. Our data show that sustained beta-AR activation induces internalization of a cardiac macromolecular complex involving VDCC and beta-arrestin 1 (beta-Arr1) into clathrin-coated vesicles. Pretreatment of myocytes with pertussis toxin prevents the internalization of VDCCs, suggesting that G(i/o) mediates this response. A peptide that selectively disrupts the interaction between Ca(V)1.2 and beta-Arr1 and tyrosine kinase inhibitors readily prevent agonist-induced VDCC internalization. These observations suggest that VDCC trafficking is mediated by G protein switching to G(i) of the beta-AR, which plays a prominent role in various cardiac pathologies associated with a hyperadrenergic state, such as hypertrophy and heart failure.

Protein kinase A, Ca2+/calmodulin-dependent kinase II, and calcineurin regulate the intracellular trafficking of myopodin between the Z-disc and the nucleus of cardiac myocytes.

Spatial and temporal resolution of intracellular signaling can be achieved by compartmentalizing transduction units. Myopodin is a dual-compartment, actin-bundling protein that shuttles between the nucleus and the Z-disc of myocytes in a differentiation- and stress-dependent fashion. Importin alpha binding and nuclear import of myopodin are regulated by serine/threonine phosphorylation-dependent binding of myopodin to 14-3-3. Here we show that in the heart myopodin forms a Z-disc signaling complex with alpha-actinin, calcineurin, Ca2+/calmodulin-dependent kinase II (CaMKII), muscle-specific A-kinase anchoring protein, and myomegalin. Phosphorylation of myopodin by protein kinase A (PKA) or CaMKII mediates 14-3-3 binding and nuclear import in myoblasts. Dephosphorylation of myopodin by calcineurin abrogates 14-3-3beta binding. Activation of PKA or inhibition of calcineurin in adult cardiac myocytes releases myopodin from the Z-disc and induces its nuclear import. The identification of myopodin as a direct target of PKA, CaMKII, and calcineurin defines a novel intracellular signaling pathway whereby changes in Z-disc dynamics may translate into compartmentalized signal transduction in the heart.

CXCR4 modulates contractility in adult cardiac myocytes.

The inflammatory response is critical to the development and progression of heart failure. Chemokines and their receptors are a distinct class of inflammatory modulators that may play a role in mediating myocardial dysfunction in heart failure. Levels of the chemokine CXCL12, also known as stromal cell-derived factor (SDF), and its receptor, CXCR4, are elevated in patients with heart failure, and we undertook this study to determine whether this chemokine system can directly affect cardiac function in the absence of leukocytes. Murine papillary muscles and adult rat cardiac myocytes treated with CXCL12, the only identified ligand of CXCR4, demonstrate blunted inotropic responses to physiologic concentrations of calcium. The negative inotropic effects on cardiac myocytes are accompanied by a proportional diminution of calcium transients. The effects are abrogated by AMD3100, a specific CXCR4 inhibitor. Overexpression of the receptor through adenoviral infection with a CXCR4 construct accentuates the negative inotropic effects of CXCL12 on cardiac myocytes during calcium stimulation. CXCR4 activation also attenuates beta-adrenergic-mediated increases in calcium mobilization and fractional shortening in cardiac myocytes. In electrophysiologic studies, CXCL12 decreases forskolin- and isoproterenol-induced voltage-gated L-type calcium channel activation. These studies demonstrate that activation of CXCR4 results in a direct negative inotropic modulation of cardiac myocyte function. The specific mechanism of action involves alterations of calcium channel activity on the membrane. The presence of functional CXCR4 on cardiac myocytes introduces a new target for treating cardiac dysfunction.

Chemokine receptors in vascular smooth muscle.

Atherosclerosis is considered to be an inflammatory disease. Chemokines are low-molecular-weight proteins that exert their effects, in part, through mediating leukocytic infiltration into the vessel wall. Recently, studies have determined that chemokines and their receptors are present, and function on other cellular components comprising the arterial wall, such as the endothelium and vascular smooth muscle. Smooth muscle cells (SMC) constitute the major cellular element of the arterial wall and are located predominantly in the arterial media. Recent studies have demonstrated that SMC possess a number of functional chemokine receptors, including CCR5, CXCR4, and a receptor for monocyte chemoattractant protein-1 (MCP-1). It is likely that SMC are increasingly recognized as potential targets for chemokines, and that these effects may influence a variety of normal and pathological processes involving SMC such as atherosclerosis and arterial injury.