Cathelicidin Antimicrobial Peptide Levels in Atherosclerosis and Myocardial Infarction in Mice and Human.

Obesity represents a worldwide health challenge, and the condition is accompanied by elevated risk of cardiovascular diseases caused by metabolic dysfunction and proinflammatory adipokines. Among those, the immune-modulatory cathelicidin antimicrobial peptide (human: CAMP; murine: CRAMP) might contribute to the interaction of the innate immune system and metabolism in these settings. We investigated systemic CAMP/CRAMP levels in experimental murine models of atherosclerosis, myocardial infarction and cardiovascular patients. Atherosclerosis was induced in low-density lipoprotein receptor-deficient (Ldlr-/-) mice by high-fat diet (HFD). C57BL/6J wild-type mice were subjected to myocardial infarction by permanent or transient left anterior descending (LAD)-ligation. Cramp gene expression in murine organs and tissues was investigated via real-time PCR. Blood samples of 234 adult individuals with or without coronary artery disease (CAD) were collected. Human and murine CAMP/CRAMP serum levels were quantified by ELISA. Atherosclerotic mice exhibited significantly increased CRAMP serum levels and induced Cramp gene expression in the spleen and liver, whereas experimental myocardial infarction substantially decreased CRAMP serum levels. Human CAMP serum quantities were not significantly affected by CAD while being correlated with leukocytes and pro-inflammatory cytokines. Our data show an influence of cathelicidin in experimental atherosclerosis, myocardial infarction, as well as in patients with CAD. Further studies are needed to elucidate the pathophysiological mechanism.

Regulation of Cathelicidin Antimicrobial Peptide (CAMP) Gene Expression by TNFα and cfDNA in Adipocytes.

Understanding the complex interactions between metabolism and the immune system ("metaflammation") is crucial for the identification of key immunomodulatory factors as potential therapeutic targets in obesity and in cardiovascular diseases. Cathelicidin antimicrobial peptide (CAMP) is an important factor of innate immunity and is expressed in adipocytes. CAMP, therefore, might play a role as an adipokine in metaflammation and adipose inflammation. TNFα, cell-free nucleic acids (cfDNA), and toll-like receptor (TLR) 9 are components of the innate immune system and are functionally active in adipose tissue. The aim of the present study was to investigate the impact of TNFα and cfDNA on CAMP expression in adipocytes. Since cfDNA acts as a physiological TLR9 agonist, we additionally investigated TLR9-mediated CAMP regulation in adipocytes and adipose tissue. CAMP gene expression in murine 3T3-L1 and human SGBS adipocytes and in murine and human adipose tissues was quantified by real-time PCR. Adipocyte inflammation was induced in vitro by TNFα and cfDNA stimulation. Serum CAMP concentrations in TLR9 knockout (KO) and in wildtype mice were quantified by ELISA. In primary adipocytes of wildtype and TLR9 KO mice, CAMP gene expression was quantified by real-time PCR. CAMP gene expression was considerably increased in 3T3-L1 and SGBS adipocytes during differentiation. TNFα significantly induced CAMP gene expression in mature adipocytes, which was effectively antagonized by inhibition of PI3K signaling. Cell-free nucleic acids (cfDNA) significantly impaired CAMP gene expression, whereas synthetic agonistic and antagonistic TLR9 ligands had no effect. CAMP and TLR9 gene expression were correlated positively in murine and human subcutaneous but not in intra-abdominal/visceral adipose tissues. Male TLR9 knockout mice exhibited lower systemic CAMP concentrations than wildtype mice. CAMP gene expression levels in primary adipocytes did not significantly differ between wildtype and TLR9 KO mice. These findings suggest a regulatory role of inflammatory mediators, such as TNFα and cfDNA, in adipocytic CAMP expression as a novel putative molecular mechanism in adipose tissue innate immunity.

Circulating Concentrations of Cathelicidin Anti-Microbial Peptide (CAMP) Are Increased during Oral Glucose Tolerance Test.

Recent investigation has revealed the significant role of Cathelicidin antimicrobial peptide (CAMP) in infection defense and innate immunity processes in adipose tissue. Meanwhile, knowledge of its regulation and functions in metabolic contexts as an adipokine remains sparce. The present study investigated the postprandial regulation of circulating CAMP levels during oral glucose tolerance tests (OGTTs). Eighty-six metabolically healthy volunteers participated in a standardized 75 g-2 h-OGTT setting. The effects of exogenous glucose, insulin, and incretins on CAMP expression in human adipocyte culture (cell-line SGBS) were studied in vitro. CAMP concentrations in blood serum samples were measured by ELISA techniques and adipocyte gene expression levels were quantified by real-time PCR. Of note, base-line CAMP serum quantities were negatively correlated with HDL cholesterol levels as well as with the anti-inflammatory adipokine adiponectin. During the 2 h following glucose ingestion, a significant rise in circulating CAMP concentrations was observed in considerable contrast to reduced quantities of fatty acid binding proteins (FABP) 2 and 4 and dipeptidyl peptidase 4 (DPP4). In SGBS adipocytes, neither differing glucose levels nor insulin or incretin treatment significantly induced CAMP mRNA levels. According to our data, glucose represents a positive postprandial regulator of systemic CAMP. This effect apparently is not mediated by the regulatory impact of glucose metabolism on adipocyte CAMP expression.

Circulating Levels of Cathelicidin Antimicrobial Peptide (CAMP) Are Affected by Oral Lipid Ingestion.

INTRODUCTION: Obesity and related diseases are among the main public health issues in the western world. They are thought to be caused by a state of chronic, low-grade inflammation. Cathelicidin antimicrobial peptide (CAMP) was recently discovered to be expressed and secreted by adipocytes. Representing a novel immunomodulatory adipokine, CAMP might play an important role in the complex interaction between metabolism and inflammation. METHODS: In a cohort of 80 volunteers, serum samples were collected prior to, and 2 h, 4 h, and 6 h after, oral lipid ingestion. CAMP, fatty acid binding proteins 2 and 4 (FABP-2/-4), and dipeptidylpeptidase-4 (DPP-4) serum concentrations were measured via ELISA. Human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were treated with free fatty acids, and gene expression levels of CAMP, FABP-4, and DPP-4 were quantified by RT-PCR. RESULTS: The mean base-line CAMP serum concentration was 55.78 ± 29.26 ng/mL, with a range of 10.77-146.24 ng/mL. Interestingly, CAMP serum levels were positively correlated with LDL cholesterol, but negatively correlated with HDL cholesterol and adiponectin. Men exhibited higher CAMP serum concentrations than women, an effect apparently linked to oral contraception in the majority of female participants. In both genders, CAMP serum concentrations significantly decreased in a stepwise manner 4 h and 6 h after oral lipid ingestion. This decline was paralleled by a rise of serum bile acid and triglyceride levels upon lipid ingestion. In human SGBS adipocytes, treatment with free fatty acids did not affect CAMP gene expression, but increased FABP-4 gene expression. CONCLUSIONS: In conclusion, systemic levels of the antimicrobial peptide and novel adipokine CAMP are significantly decreased upon oral lipid ingestion. While this decline might be linked to the simultaneous increase in bile acids, the underlying mechanisms remain to be elucidated. Furthermore, CAMP might indicate a putative novel cardiovascular biomarker of both inflammatory and metabolic relevance in metaflammation and adipose inflammation.

Impact of oral lipid and glucose tolerance tests on the postprandial concentrations of angiopoietin-like proteins (Angptl) 3 and 4.

BACKGROUND: The postprandial regulation of angiopoietin-like proteins (Angptls) and their expression in adipocytes is poorly characterized. OBJECTIVE: Circulating Angptl3 and 4 were analyzed in healthy individuals undergoing either an oral lipid tolerance test (OLTT; n = 98) or an oral glucose tolerance test (OGTT; n = 99). Venous blood was drawn after 0, 2, 4, and 6 h during OLTT and after 0, 1, and 2 h during OGTT. Anthropometric and laboratory parameters were assessed and concentrations of Angptls were quantified by enzyme-linked immunosorbent assay. Angptl gene expression in 3T3-L1 adipocytes and in murine adipose tissues and cellular fractions was analyzed by quantitative real-time PCR. RESULTS: Angptl3 concentrations significantly decreased while Angptl4 levels continuously increased during OLTT. Both proteins remained unaffected during OGTT. Angptl3 and Angptl4 were expressed in murine subcutaneous and visceral AT with higher mRNA levels in mature adipocytes when compared to the stroma-vascular cell fraction. Both proteins were strongly induced during 3T3-L1 adipocyte differentiation and they were unresponsive to glucose in mature fat cells. Adipocyte Angptl3 (but not Angptl4) mRNA expression was inhibited by the polyunsaturated fatty acids arachidonic acid and docosahexaenoic acid, whereas nine types of dietary fatty acids remained without any effect. CONCLUSIONS: There is evidence of short-time regulation of Angptl3/4 levels upon metabolic stress. Angptl4 expression is high and Angptl3 expression is low in AT and restricted mainly to mature adipocytes without any differences concerning fat compartments. Whereas dietary fatty acids and glucose are without any effect, omega-3/-6-polyunsaturated fatty acids inhibited Anptl3 expression in adipocytes.

Evidence of a Muscle-Brain Axis by Quantification of the Neurotrophic Myokine METRNL (Meteorin-Like Protein) in Human Cerebrospinal Fluid and Serum.

Data on the quantification of the potentially neurotrophic adipo-myokine METRNL (Meteorin-like protein) in human cerebrospinal fluid (CSF) are lacking and migration of this secreted protein across the blood-brain barrier (BBB) is uncertain. In the present pilot study, METRNL concentrations were quantified by ELISA in paired serum and CSF samples of 260 patients (107 males, 153 females) undergoing neurological evaluation. METRNL was abundant in serum (801.2 ± 378.3 pg/mL) and CSF (1007.2 ± 624.2 pg/mL) with a CSF/serum ratio of 1.4 ± 0.8. Serum METRNL levels were significantly correlated (rho = +0.521) to those in CSF. CSF METRNL concentrations were significantly correlated (rho = +0.480) with albumin CSF/serum ratios. The CSF/serum ratios of METRNL and albumin were positively correlated in Reibergram analysis (rho = 0.498), indicating that raising CSF concentrations of METRNL are mediated by increasing BBB dysfunction. The CSF concentrations of METRNL strongly increased in a stepwise manner along with increasing BBB dysfunction from grade 0 to grade 3 and with rising CSF cell count. CSF/serum ratio of METRNL also increased from grade 0 (1.2 ± 0.7) to grade 3 (3.0 ± 0.2). Furthermore, CSF levels were positively correlated with age. In conclusion, METRNL is a secreted and neurotrophic myokine that crosses over the BBB. CSF concentrations of METRNL increase with BBB dysfunction.

The adipokine C1q/TNF-related protein-3 (CTRP-3) inhibits Toll-like receptor (TLR)-induced expression of Cathelicidin antimicrobial peptide (CAMP) in adipocytes.

BACKGROUND AND AIM: CAMP (Cathelicidin antimicrobial peptide) expression in adipocytes is regulated by Toll-like receptor (TLR) agonists. Secreted adipokines such as CTRP-3 have been suggested to participate in innate immune signaling in adipose tissue (AT). This study investigates whether TLR-induced CAMP expression in adipocytes is antagonized by CTRP-3. METHODS: 3T3-L1 adipocytes were co-stimulated with TLR agonists (LPS, MALP-2, Pam3CSK4, pI:C) and recombinant CTRP-3. In a SIRS model, C57BL/6 wild-type mice were intraperitoneally (ip) injected with recombinant CTRP-3 prior to LPS. CAMP expression was analyzed by real-time PCR in AT of wild-type mice and in AT and primary adipocytes from transgenic mice lacking adipocyte CTRP-3 expression. Comparative transcriptome analysis by RNA seq. was applied in CTRP-3 KO adipocytes. RESULTS: In vitro, CTRP-3 antagonized TLR4- and TLR1/2-induced CAMP expression in adipocytes whereas TLR3- and TLR2/6-mediated induction of CAMP was not affected. in vivo, application of exogenous CTRP-3 dose-dependently antagonized LPS-induced CAMP expression in intra-abdominal AT. CAMP expression in total AT and in primary adipocytes of subcutaneous and intra-abdominal AT did not differ between wild-type mice and transgenic mice lacking adipocyte CTRP-3 expression. CONCLUSIONS: The study suggests a hypothetical role of CAMP in host defense not only against Gram-positive bacteria sensed by TLR1/2 and TLR2/6 but also against Gram-negative bacteria sensed by TLR4 and potentially against viruses sensed by TLR3. The machinery of TLR-mediated pro-inflammatory activation of the CAMP gene in adipocytes seems to be partly modulated by secreted adipokines belonging to the growing family of C1q/TNF-related proteins such as CTRP-3.

Regulation of CAMP (cathelicidin antimicrobial peptide) expression in adipocytes by TLR 2 and 4.

Recent data argue for a pro-inflammatory role of CAMP (cathelicidin antimicrobial peptide) in adipocytes and adipose tissue (AT) and for regulatory circuits involving TLRs. In order to investigate regulatory effects of TLR2 and TLR4, 3T3-L1 adipocytes were stimulated with TLR2 agonistic lipopeptide MALP-2 and with TLR4 agonist LPS in presence or absence of signal transduction inhibitors. CAMP gene expression was analysed by quantitative real-time PCR in adipocytes and in murine AT compartments and cellular subfractions. CAMP expression was higher in gonadal than in subcutaneous AT and there was a gender-specific effect with higher levels in males. Adipocytes had higher CAMP expression than the stroma-vascular cell (SVC) fraction. MALP-2 up-regulated CAMP expression significantly, mediated by STAT3 and PI3K and potentially (non-significant trend) by NF-κB and MAPK, but not by raf-activated MEK-1/-2. Moreover, LPS proved to act as a potent inducer of CAMP via NF-κB, PI3K and STAT3, whereas specific inhibition of MAPK and MEK-1/-2 had no effect. In conclusion, activation of TLR2 and TLR4 by classical ligands up-regulates adipocyte CAMP expression involving classical signal transduction elements. These might represent future drug targets for pharmacological modulation of CAMP expression in adipocytes, especially in the context of metabolic and infectious diseases.

Systematic Quantification of Neurotrophic Adipokines RBP4, PEDF, and Clusterin in Human Cerebrospinal Fluid and Serum.

CONTEXT: Data on the presence/quantification of the neurotrophic adipokines retinol-binding protein-4 (RBP4), clusterin, and pigment epithelium-derived factor (PEDF) in human cerebrospinal fluid (CSF) are scarce and migration of these adipokines across of the blood-brain barrier (BBB) is uncertain. OBJECTIVE: This work aimed to quantify RBP4, PEDF, and clusterin in paired serum and CSF samples of patients undergoing neurological evaluation. METHODS: A total of 268 patients (109 male, 159 female) were included. Adipokine serum and CSF concentrations were measured by enzyme-linked immunosorbent assay in duplicate. RESULTS: RBP4 was abundant in serum (mean, 31.9 ±â€…24.2 µg/mL). The serum concentrations were approximately 145 times higher than in CSF (CSF to serum RBP4 ratio, 8.2 ±â€…4.3 × 10-3). PEDF was detectable in serum (mean, 30.2 ±â€…11.7 µg/mL) and concentrations were approximately 25 times higher than in CSF (CSF to serum PEDF ratio, 42.3 ±â€…15.6 × 10-3). Clusterin serum concentrations were abundant with mean levels of 346.0 ±â€…114.6 µg/mL, which were approximately 40 times higher than CSF levels (CSF to serum clusterin ratio, 29.6 ±â€…23.4 × 10-3). RBP4 and PEDF serum levels correlated positively with CSF levels, which were increased in overweight/obese patients and in type 2 diabetic patients. The CSF concentrations of all 3 adipokines increased with BBB dysfunction. RBP4 in CSF correlated positively with inflammatory parameters. In detail, only RBP4 showed the kinetics and associations that are mandatory for a putative mediator of the fat-brain axis. CONCLUSION: RBP4, PEDF, and clusterin are permeable to the BBB and increase with the measure of BBB dysfunction. RBP4 represents an inflammatory neurotrophic adipokine and is a promising mediator of the fat-brain axis.