David Deng

Novel Role for the Potent Endogenous Inotrope Apelin in Human Cardiac Dysfunction

Background—Apelin is among the most potent stimulators of cardiac contractility known. However, no physiological or pathological role for apelin–angiotensin receptor-like 1 (APJ) signaling has ever been described. Methods and Results—We performed transcriptional profiling using a spotted cDNA microarray with 12 814 unique clones on paired samples of left ventricle obtained before and after placement of a left ventricular assist device in 11 patients. The significance analysis of microarrays and a novel rank consistency score designed to exploit the paired structure of the data confirmed that natriuretic peptides were among the most significantly downregulated genes after offloading. The most significantly upregulated gene was the G-protein–coupled receptor APJ, the specific receptor for apelin. We demonstrate here using immunoassay and immunohistochemical techniques that apelin is localized primarily in the endothelium of the coronary arteries and is found at a higher concentration in cardiac tissue after mechanical offloading. These findings imply an important paracrine signaling pathway in the heart. We additionally extend the clinical significance of this work by reporting for the first time circulating human apelin levels and demonstrating increases in the plasma level of apelin in patients with left ventricular dysfunction. Conclusions—The apelin-APJ signaling pathway emerges as an important novel mediator of cardiovascular control.

Differences in Vascular Bed Disease Susceptibility Reflect Differences in Gene Expression Response to Atherogenic Stimuli

Atherosclerosis occurs predominantly in arteries and only rarely in veins. The goal of this study was to test whether differences in the molecular responses of venous and arterial endothelial cells (ECs) to atherosclerotic stimuli might contribute to vascular bed differences in susceptibility to atherosclerosis. We compared gene expression profiles of primary cultured ECs from human saphenous vein (SVEC) and coronary artery (CAEC) exposed to atherogenic stimuli. In addition to identifying differentially expressed genes, we applied statistical analysis of gene ontology and pathway annotation terms to identify signaling differences related to cell type and stimulus. Differential gene expression of untreated venous and arterial endothelial cells yielded 285 genes more highly expressed in untreated SVEC (P<0.005 and fold change >1.5). These genes represented various atherosclerosis-related pathways including responses to proliferation, oxidoreductase activity, antiinflammatory responses, cell growth, and hemostasis functions. Moreover, stimulation with oxidized LDL induced dramatically greater gene expression responses in CAEC compared with SVEC, relating to adhesion, proliferation, and apoptosis pathways. In contrast, interleukin 1&bgr; and tumor necrosis factor &agr; activated similar gene expression responses in both CAEC and SVEC. The differences in functional response and gene expression were further validated by an in vitro proliferation assay and in vivo immunostaining of &agr;&bgr;-crystallin protein. Our results strongly suggest that different inherent gene expression programs in arterial versus venous endothelial cells contribute to differences in atherosclerotic disease susceptibility.

Molecular Signatures Determining Coronary Artery and Saphenous Vein Smooth Muscle Cell Phenotypes. Distinct Responses to Stimuli

Objective—Phenotypic differences between vascular smooth muscle cell (VSMC) subtypes lead to diverse pathological processes including atherosclerosis, postangioplasty restenosis and vein graft disease. To better understand the molecular mechanisms underlying functional differences among distinct SMC subtypes, we compared gene expression profiles and functional responses to oxidized low-density lipoprotein (OxLDL) and platelet-derived growth factor (PDGF) between cultured SMCs from human coronary artery (CASM) and saphenous vein (SVSM). Methods and Results—OxLDL and PDGF elicited markedly different functional responses and expression profiles between the 2 SMC subtypes. In CASM, OxLDL inhibited cell proliferation and migration and modified gene expression of chemokines (CXCL10, CXCL11 and CXCL12), proinflammatory cytokines (IL-1, IL-6, and IL-18), insulin-like growth factor binding proteins (IGFBPs), and both endothelial and smooth muscle marker genes. In SVSM, OxLDL promoted proliferation partially via IGF1 signaling, activated NF-&kgr;B and phosphatidylinositol signaling pathways, and upregulated prostaglandin (PG) receptors and synthases. In untreated cells, &agr;-chemokines, proinflammatory cytokines, and genes associated with apoptosis, inflammation, and lipid biosynthesis were higher in CASM, whereas some &bgr;-chemokines, metalloproteinase inhibitors, and IGFBPs were higher in SVSM. Interestingly, the basal expression levels of these genes seemed closely related to their responses to OxLDL and PDGF. In summary, our results suggest dramatic differences in gene expression patterns and functional responses to OxLDL and PDGF between venous and arterial SMCs, with venous SMCs having stronger proliferative/migratory responses to stimuli but also higher expression of atheroprotective genes at baseline. Conclusions—These results reveal molecular signatures that define the distinct phenotypes characteristics of coronary artery and saphenous vein SMC subtypes.

PROCAM Study: risk prediction for myocardial infarction using microfluidic high-density lipoprotein (HDL) subfractionation is independent of HDL cholesterol.

Abstract Background: High-density lipoprotein (HDL) subfractions are among the new emerging risk factors for atherosclerosis. In particular, HDL 2b has been shown to be linked to cardiovascular risk. This study uses a novel microfluidics-based method to establish HDL 2b clinical utility using samples from the Prospective Cardiovascular Muenster (PROCAM) Study. Methods: Method performance was established by measuring accuracy, precision, linearity and inter-site precision. Serum samples from 503 individuals collected in the context of the PROCAM study were analyzed by electrophoresis on a microfluidics system. Of these, 251 were male survivors of myocardial infarction (cases), while 252 individuals were matched healthy controls. HDL cholesterol, HDL 2b concentration and HDL 2b percentage were analyzed. Results: This novel method showed satisfactory assay performance with an inter-site coefficient of variance of <10% for HDL 2b percentage. Parallel patient testing on 52 samples between two sites resulted in a correlation coefficient of r=0.95. Significant differences were observed in the HDL 2b subfraction between cases and controls independent of other risk factors. Including HDL 2b percentage in logistic regression reduced the number of false positives from 64 to 39 and the number of false negative cases from 48 to 45, in the context of this study. Conclusions: The novel method showed satisfactory assay performance in addition to drastically reduced analysis times and improved ease of use as compared to other methods. Clinical utility of HDL 2b was demonstrated supporting the findings of previous studies. Clin Chem Lab Med 2008;46:490–8.

Adrenergic Receptors in Individual Ventricular Myocytes

Rationale: It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), &bgr;1, &bgr;2, &bgr;3, &agr;1A, and &agr;1B. The &bgr;1 and &bgr;2 are thought to be the dominant myocyte ARs. Objective: Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results: We studied ventricular myocytes from wild-type mice, mice with &agr;1A and &agr;1B knockin reporters, and &bgr;1 and &bgr;2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal–regulated kinase and phospholamban), and contraction. We found that the &bgr;1 and &agr;1B were present in all myocytes. The &agr;1A was present in 60%, with high levels in 20%. The &bgr;2 and &bgr;3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total &bgr;-ARs were &bgr;2 and 20% were &bgr;3, both mainly in nonmyocytes. Conclusion: The dominant ventricular myocyte ARs present in all cells are the &bgr;1 and &agr;1B. The &bgr;2 and &bgr;3 are mostly absent in myocytes but are abundant in nonmyocytes. The &agr;1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with &bgr;1 and &agr;1B only; 60% that also have the &agr;1A; and 5% each that also have the &bgr;2 or &bgr;3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms.Rationale: It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), β1, β2, β3, α1A, and α1B. The β1 and β2 are thought to be the dominant myocyte ARs. Objective: Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results: We studied ventricular myocytes from wild-type mice, mice with α1A and α1B knockin reporters, and β1 and β2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal–regulated kinase and phospholamban), and contraction. We found that the β1 and α1B were present in all myocytes. The α1A was present in 60%, with high levels in 20%. The β2 and β3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total β-ARs were β2 and 20% were β3, both mainly in nonmyocytes. Conclusion: The dominant ventricular myocyte ARs present in all cells are the β1 and α1B. The β2 and β3 are mostly absent in myocytes but are abundant in nonmyocytes. The α1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with β1 and α1B only; 60% that also have the α1A; and 5% each that also have the β2 or β3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms. # Novelty and Significance {#article-title-74}

Adrenergic Receptors in Individual Ventricular MyocytesNovelty and Significance

Rationale: It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), &bgr;1, &bgr;2, &bgr;3, &agr;1A, and &agr;1B. The &bgr;1 and &bgr;2 are thought to be the dominant myocyte ARs. Objective: Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results: We studied ventricular myocytes from wild-type mice, mice with &agr;1A and &agr;1B knockin reporters, and &bgr;1 and &bgr;2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal–regulated kinase and phospholamban), and contraction. We found that the &bgr;1 and &agr;1B were present in all myocytes. The &agr;1A was present in 60%, with high levels in 20%. The &bgr;2 and &bgr;3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total &bgr;-ARs were &bgr;2 and 20% were &bgr;3, both mainly in nonmyocytes. Conclusion: The dominant ventricular myocyte ARs present in all cells are the &bgr;1 and &agr;1B. The &bgr;2 and &bgr;3 are mostly absent in myocytes but are abundant in nonmyocytes. The &agr;1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with &bgr;1 and &agr;1B only; 60% that also have the &agr;1A; and 5% each that also have the &bgr;2 or &bgr;3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms.Rationale: It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), β1, β2, β3, α1A, and α1B. The β1 and β2 are thought to be the dominant myocyte ARs. Objective: Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results: We studied ventricular myocytes from wild-type mice, mice with α1A and α1B knockin reporters, and β1 and β2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal–regulated kinase and phospholamban), and contraction. We found that the β1 and α1B were present in all myocytes. The α1A was present in 60%, with high levels in 20%. The β2 and β3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total β-ARs were β2 and 20% were β3, both mainly in nonmyocytes. Conclusion: The dominant ventricular myocyte ARs present in all cells are the β1 and α1B. The β2 and β3 are mostly absent in myocytes but are abundant in nonmyocytes. The α1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with β1 and α1B only; 60% that also have the α1A; and 5% each that also have the β2 or β3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms. # Novelty and Significance {#article-title-74}

Adrenergic Receptors in Individual Ventricular MyocytesNovelty and Significance: The Beta-1 and Alpha-1B Are in All Cells, the Alpha-1A Is in a Subpopulation, and the Beta-2 and Beta-3 Are Mostly Absent

Rationale: It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), &bgr;1, &bgr;2, &bgr;3, &agr;1A, and &agr;1B. The &bgr;1 and &bgr;2 are thought to be the dominant myocyte ARs. Objective: Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results: We studied ventricular myocytes from wild-type mice, mice with &agr;1A and &agr;1B knockin reporters, and &bgr;1 and &bgr;2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal–regulated kinase and phospholamban), and contraction. We found that the &bgr;1 and &agr;1B were present in all myocytes. The &agr;1A was present in 60%, with high levels in 20%. The &bgr;2 and &bgr;3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total &bgr;-ARs were &bgr;2 and 20% were &bgr;3, both mainly in nonmyocytes. Conclusion: The dominant ventricular myocyte ARs present in all cells are the &bgr;1 and &agr;1B. The &bgr;2 and &bgr;3 are mostly absent in myocytes but are abundant in nonmyocytes. The &agr;1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with &bgr;1 and &agr;1B only; 60% that also have the &agr;1A; and 5% each that also have the &bgr;2 or &bgr;3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms.Rationale: It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), β1, β2, β3, α1A, and α1B. The β1 and β2 are thought to be the dominant myocyte ARs. Objective: Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results: We studied ventricular myocytes from wild-type mice, mice with α1A and α1B knockin reporters, and β1 and β2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal–regulated kinase and phospholamban), and contraction. We found that the β1 and α1B were present in all myocytes. The α1A was present in 60%, with high levels in 20%. The β2 and β3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total β-ARs were β2 and 20% were β3, both mainly in nonmyocytes. Conclusion: The dominant ventricular myocyte ARs present in all cells are the β1 and α1B. The β2 and β3 are mostly absent in myocytes but are abundant in nonmyocytes. The α1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with β1 and α1B only; 60% that also have the α1A; and 5% each that also have the β2 or β3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms. # Novelty and Significance {#article-title-74}