Are primed polymorphonuclear leukocytes contributors to the high heparanase levels in hemodialysis patients?

2008 ◽  
Vol 294 (2) ◽  
pp. H651-H658 ◽  
Author(s):  
Meital Cohen-Mazor ◽  
Shifra Sela ◽  
Rafi Mazor ◽  
Neta Ilan ◽  
Israel Vlodavsky ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Polymorphonuclear Leukocytes ◽  
Atherosclerotic Lesion ◽  
Cardiovascular Complications ◽  
Chronic Hemodialysis ◽  
Dialysis Session ◽  
Rt Pcr ◽  
Protein Levels ◽  
Atherosclerotic Lesions ◽  
Atherosclerotic Process

Patients on chronic hemodialysis (HD) are at high risk for developing atherosclerosis and cardiovascular complications. Heparanase, an endoglycosidase that cleaves heparan sulfate (HS) side chains of proteoglycans, is involved in extracellular matrix degradation and, as such, may be involved in the atherosclerotic lesion progression. We hypothesize that heparanase is elevated in HD patients, partly due to its release from primed circulating polymorphonuclear leukocytes (PMNLs), undergoing degranulation. Priming of PMNLs was assessed by levels of CD11b and the rate of superoxide release. Heparanase mRNA expression in PMNLs was determined by RT-PCR. PMNL and plasma levels of heparanase were determined by immunoblotting, immunofluorescence, and flow cytometry analyses. The levels of soluble HS in plasma were measured by a competition ELISA. This study shows that PMNLs isolated from HD patients have higher mRNA and protein levels of heparanase compared with normal control (NC) subjects and that heparanase levels correlate positively with PMNL priming. Plasma levels of heparanase were higher in HD patients than in NC subjects and were further elevated after the dialysis session. In addition, heparanase expression inversely correlates with plasma HS levels. A pronounced expression of heparanase was found in human atherosclerotic lesions. The increased heparanase activity in the blood of HD patients results at least in part from the degranulation of primed PMNLs and may contribute to the acceleration of the atherosclerotic process. Our findings highlight primed PMNLs as a possible source for the increased heparanase in HD patients, posing heparanase as a new risk factor for cardiovascular complications and atherosclerosis.

scholarly journals Triage of Patients Suspected of COVID-19 in Chronic Hemodialysis: Eosinophil Count Differentiates Low and High Suspicion of COVID-19

2020 ◽  
Vol 10 (1) ◽  
pp. 4
Author(s):  
Romain Vial ◽  
Marion Gully ◽  
Mickael Bobot ◽  
Violaine Scarfoglière ◽  
Philippe Brunet ◽  
...  
Keyword(s):  
Predictive Value ◽  
Eosinophil Count ◽  
Characteristic Curve ◽  
Final Analysis ◽  
Hemodialysis Patients ◽  
Chronic Hemodialysis ◽  
Screening Methods ◽  
Dialysis Session ◽  
Diagnostic Study ◽  
Rt Pcr

Background: Daily management to shield chronic dialysis patients from SARS-CoV-2 contamination makes patient care cumbersome. There are no screening methods to date and a molecular biology platform is essential to perform RT-PCR for SARS-CoV-2; however, accessibility remains poor. Our goal was to assess whether the tools routinely used to monitor our hemodialysis patients could represent reliable and quickly accessible diagnostic indicators to improve the management of our hemodialysis patients in this pandemic environment. Methods: In this prospective observational diagnostic study, we recruited patients from La Conception hospital. Patients were eligible for inclusion if suspected of SARS-CoV-2 infection when arriving at our center for a dialysis session between March 12th and April 24th 2020. They were included if both RT-PCR result for SARS-CoV-2 and cell blood count on the day that infection was suspected were available. We calculated the area under the curve (AUC) of the receiver operating characteristic curve. Results: 37 patients were included in the final analysis, of which 16 (43.2%) were COVID-19 positive. For the day of suspected COVID-19, total leukocytes were significantly lower in the COVID-19 positive group (4.1 vs. 7.4 G/L, p = 0.0072) and were characterized by lower neutrophils (2.7 vs. 5.1 G/L, p = 0.021) and eosinophils (0.01 vs. 0.15 G/L, p = 0.0003). Eosinophil count below 0.045 G/L identified SARS-CoV-2 infection with AUC of 0.9 [95% CI 0.81—1] (p < 0.0001), sensitivity of 82%, specificity of 86%, a positive predictive value of 82%, a negative predictive value of 86% and a likelihood ratio of 6.04. Conclusions: Eosinophil count enables rapid routine screening of symptomatic chronic hemodialysis patients suspected of being COVID-19 within a range of low or high probability.

P6195Nanoliposomal anti-PCSK9 vaccine induces long-term and safe protection against atherosclerosis in C57BL/6 mouse

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
A A Momtazi-Borojeni ◽  
M R Jaafari ◽  
A A Badiee ◽  
M Banach ◽  
A A Sahebkar
Keyword(s):  
Lipid Profile ◽  
Plasma Levels ◽  
Atherosclerotic Lesion ◽  
Atherogenic Diet ◽  
Negative Control ◽  
Th2 Cells ◽  
Protective Effects ◽  
Lipid Film ◽  
Protein Levels

Abstract Background Proprotein convertase subtilisin/kexin 9 (PCSK9) can increase plasma levels of low-density lipoprotein (LDL) cholesterol (LDL-C) through reducing protein levels of liver LDL receptor (LDLR). PCSK9 inhibition has been found to efficiently alleviate hypercholesterolemia. Purpose In the present study, we prepared nanoliposomal anti-PCSK9 vaccine and evaluated its protective effects against hypercholesterolemia and atherosclerotic plaque formation in C57BL/6 mice fed on atherogenic diet. Methods Nanoliposome formulation containing 1,2-Dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) and cholesterol (Chol) were prepared using the lipid-film hydration method, and used as the vaccine delivery system. Immunogenic peptide called Immunogenic Fused PCSK9-Tetanus (IFPT) was exhibited on the surface of nanoliposome carriers by using DSPE-PEG-Maleimide lipid (L-IFPT) and adsorbed to Alum adjuvant (L-IFPTA+). The prepared vaccine formulations IFPT, L-IFPTA+, IFPTA+, IFPT, and empty liposome as the negative control were subcutaneously administrated four times with a bi-weekly interval in C57BL/6 mice fed on atherogenic diet. Protein levels of liver LDLR, lipid profile, plasma levels of anti-PCSK9 antibody, plasma concentration of PCSK9 protein, and effect of anti-PCSK9 antibody on PCSK9-LDLR interaction were measured in the vaccinated mice. Aortic arch atherosclerotic lesions were studied using hematoxylin and eosin staining. To determine inflammatory response, splenic CD4+producing IL-4 and IFN-γ were evaluated using flow cytometry analysis. Results Among formulated vaccines, the L-IFPTA+vaccine could generate the highest IgG anti-PCSK9 antibody in the vaccinated hypercholesterolemic mice. The generated anti-PCSK9 antibodies inhibited interaction of PCSK9 with LDLR through targeting plasma PCSK9, whereby protein levels of liver LDLR was elevated in the vaccinated mice. As reveled by lipid profile analysis, liposomal vaccines, with more extent the L-IFPTA+vaccine, could significantly decrease plasma TC and LDL-C in hypercholesterolemic mice. Of note, L-IFPTA+ vaccine could reduce size and severity of atherosclerotic lesion in the aorta arch. Importantly, long-term (48 weeks) follow-up of hypercholesterolemic vaccinated mice revealed that L-IFPTA+vaccine could induce a long-lasting humoral immune response against plasma PCSK9, which was accompanied with a significant decrease of TC and LDL-C (Figure). Additionally, Anti-inflammatory Th2 cells and IL-4 cytokine were significantly elevated in splenic cells isolated from hypercholesterolemic vaccinated mice. Figure 1 Conclusions L-IFPTA+vaccine can promote long-lasting, functional and safe anti-PCSK9 antibodies in hypercholesterolemic C57BL/6 mice, revealing long-term protective effect against hypercholesterolemia and atherosclerosis.

scholarly journals Triage of Patients Suspected of COVID-19 in Chronic Hemodialysis: Eosinophil Count Differentiates Low and High Suspicion of COVID-19

2020 ◽  
Author(s):  
Romain Vial ◽  
Marion Gully ◽  
Mickael Bobot ◽  
Violaine Scarfoglière ◽  
Philippe Brunet ◽  
...  
Keyword(s):  
Predictive Value ◽  
Eosinophil Count ◽  
Characteristic Curve ◽  
Final Analysis ◽  
Hemodialysis Patients ◽  
Chronic Hemodialysis ◽  
Screening Methods ◽  
Dialysis Session ◽  
Diagnostic Study ◽  
Rt Pcr

Abstract Background: Daily management to shield chronic dialysis patients from SARS-CoV-2 contamination makes patient care cumbersome. There are no screening methods to date and a molecular biology platform is essential to perform RT-PCR for SARS-CoV-2; however, accessibility remains poor. Our goal was to assess whether the tools routinely used to monitor our hemodialysis patients could represent reliable and quickly accessible diagnostic indicators to improve the management of our hemodialysis patients in this pandemic environment.Methods: In this prospective observational diagnostic study, we recruited patients from La Conception hospital. Patients were eligible for inclusion if suspected of SARS-CoV-2 infection when arriving at our center for a dialysis session between March 12th and April 24th 2020. They were included if both RT-PCR result for SARS-CoV-2 and cell blood count on the day that infection was suspected were available. We calculated the area under the curve (AUC) of the receiver operating characteristic curve.Results: 37 patients were included in the final analysis, of which 16 (43.2%) were COVID-19 positive. For the day of suspected COVID-19, total leukocytes were significantly lower in the COVID-19 positive group (4.1 vs 7.4 G/L, p=0.0072) and were characterized by lower neutrophils (2.7 vs 5.1 G/L, p=0.021) and eosinophils (0.01 vs 0.15 G/L, p=0.0003). Eosinophil count below 0.045 G/L identified SARS-CoV-2 infection with AUC of 0.9 [95% CI 0.81-1] (p<0.0001), sensitivity of 82%, specificity of 86%, a positive predictive value of 82%, a negative predictive value of 86% and a likelihood ratio of 6.04.Conclusions :Eosinophil count enables rapid routine screening of chronic hemodialysis patients suspected of being COVID-19 within a range of low or high probability.

scholarly journals Hemodynamic Effects of Endothelin-1 and Big Endothelin-1 in Chronic Hemodialysis Patients

1999 ◽  
Vol 10 (5) ◽  
pp. 1037-1044
Author(s):  
ASTRID OTTOSSON-SEEBERGER ◽  
GUNVOR AHLBORG ◽  
ANETTE HEMSÉN ◽  
JAN M. LUNDBERG ◽  
ANDERS ALVESTRAND
Keyword(s):  
Heart Disease ◽  
Ischemic Heart Disease ◽  
Plasma Levels ◽  
Plasma Concentrations ◽  
Fold Increase ◽  
Cardiovascular Complications ◽  
Chronic Hemodialysis ◽  
Ischemic Heart ◽  
Endothelin 1 ◽  
Healthy Humans

Abstract. Increased plasma concentrations of endothelin-1 (ET-1) and big endothelin-1 (big ET-1) have been reported in patients with end-stage renal failure (ESRD). In the present study, which included hemodialysis (HD) patients with (n = 21) and without (n = 32) ischemic heart disease, the putative association between plasma levels of ET-1 and big ET-1 and ischemic heart disease and the influence of the dialysis procedure on ET concentrations was investigated. This study also examined in an additional five HD patients without cardiac disease whether intravenously infused ET-1 and big ET-1 (0.2, 1, and 4 pmol/kg per min, each dose for 20 min) preserve their vasoactive potency and whether exogenous big ET-1, which in healthy humans is converted in the kidney, is still converted to ET-1 in ESRD. HD patients with ischemic heart disease demonstrated higher plasma levels of ET-1 and big ET-1 than HD patients without this disorder, and HD reduced plasma ET-1 and big ET-1 concentrations. In HD patients, the big ET-1 infusion, resulting in a 1.5-fold increase in plasma ET-1, caused a more marked and prolonged rise in mean arterial BP than ET-1 (20% versus 13%, P = 0.0001) and a slightly smaller but more prolonged decrease in estimated splanchnic blood flow than ET-1 (37% versus 44%, P = 0.02). Furthermore, big ET-1 lowered heart rate by 9% (P = 0.01) but ET-1 did not. Plasma half-lives of ET-1 and big ET-1 were longer in HD patients than in healthy humans. Thus, ET-1 and big ET-1 preserve their vasoactive potency, and circulating big ET-1 is still converted to active ET-1 in ESRD. Consequently, the increased plasma levels of ET-1 and big ET-1 noted in HD patients, especially in patients with ischemic heart disease, might play a role in the development of uremic cardiovascular complications.

scholarly journals The Initial Human Atherosclerotic Lesion and Lipoprotein Modification—A Deep Connection

2021 ◽  
Vol 22 (21) ◽  
pp. 11488
Author(s):  
Michael Torzewski
Keyword(s):  
Plasma Levels ◽  
Foam Cell ◽  
Hydrolytic Enzymes ◽  
Cell Formation ◽  
Clinical Manifestations ◽  
Atherosclerotic Lesion ◽  
Foam Cell Formation ◽  
Lysosomal Storage ◽  
Atherosclerotic Lesions ◽  
Micro Rnas

Atherosclerosis research typically focuses on the evolution of intermediate or advanced atherosclerotic lesions rather than on prelesional stages of atherogenesis. Yet these early events may provide decisive leads on the triggers of the pathologic process, before lesions become clinically overt. Thereby, it is mandatory to consider extracellular lipoprotein deposition at this stage as the prerequisite of foam cell formation leading to a remarkable accumulation of LDL (Low Density Lipoproteins). As progression of atherosclerosis displays the characteristic features of a chronic inflammatory process on the one hand and native LDL lacks inflammatory properties on the other hand, the lipoprotein must undergo biochemical modification to become atherogenic. During the last 25 years, evidence was accumulated in support of a different concept on atherogenesis proposing that modification of native LDL occurs through the action of ubiquitous hydrolytic enzymes (enzymatically modified LDL or eLDL) rather than oxidation and contending that the physiological events leading to macrophage uptake and reverse transport of eLDL first occur without inflammation (initiation with reversion). Preventing or reversing initial atherosclerotic lesions would avoid the later stages and therefore prevent clinical manifestations. This concept is in accordance with the response to retention hypothesis directly supporting the strategy of lowering plasma levels of atherogenic lipoproteins as the most successful therapy for atherosclerosis and its sequelae. Apart from but unquestionable closely related to this concept, there are several other hypotheses on atherosclerotic lesion initiation favoring an initiating role of the immune system (‘vascular-associated lymphoid tissue’ (VALT)), defining foam cell formation as a variant of lysosomal storage disease, relating to the concept of the inflammasome with crystalline cholesterol and/or mitochondrial DAMPs (damage-associated molecular patterns) being mandatory in driving arterial inflammation and, last but not least, pointing to miRNAs (micro RNAs) as pivotal players. However, direct anti-inflammatory therapies may prove successful as adjuvant components but will likely never be used in the absence of strategies to lower plasma levels of atherogenic lipoproteins, the key point of the perception that atherosclerosis is not simply an inevitable result of senescence. In particular, given the importance of chemical modifications for lipoprotein atherogenicity, regulation of the enzymes involved might be a tempting target for pharmacological research.

scholarly journals Endonuclease V Regulates Atherosclerosis Through C‐C Motif Chemokine Ligand 2‐Mediated Monocyte Infiltration

2021 ◽  
Author(s):  
Xiang Yi Kong ◽  
Camilla Huse ◽  
Kuan Yang ◽  
Jonas Øgaard ◽  
Natalia Berges ◽  
...  
Keyword(s):  
Immune Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Atherosclerotic Lesion ◽  
Plaque Burden ◽  
Enzyme Activity Assay ◽  
Endonuclease V ◽  
Atherosclerotic Lesions ◽  
Chemokine Ligand ◽  
Atherosclerotic Process ◽  
Underlying Mechanisms

Background In cardiovascular diseases, atherosclerotic disorder are the most frequent and important with respect to morbidity and mortality. Inflammation mediated by immune cells is central in all parts of the atherosclerotic progress, and further understanding of the underlying mechanisms is needed. Growing evidence suggests that deamination of adenosine‐to‐inosine in RNA is crucial for a correct immune response; nevertheless, the role of adenosine‐to‐inosine RNA editing in atherogenesis has barely been studied. Several proteins have affinity for inosines in RNA, one being ENDOV (endonuclease V), which binds and cleaves RNA at inosines. Data on ENDOV in atherosclerosis are lacking. Methods and Results Quantitative polymerase chain reaction on ENDOV mRNA showed an increased level in human carotid atherosclerotic plaques compared with control veins. Inosine‐ribonuclease activity as measured by an enzyme activity assay is detected in immune cells relevant for the atherosclerotic process. Abolishing EndoV in atherogenic apolipoprotein E‐deficient ( ApoE −/− ) mice reduces the atherosclerotic plaque burden, both in size and lipid content. In addition, in a brain stroke model, mice without ENDOV suffer less damage than control mice. Finally, lack of EndoV reduces the recruitment of monocytes to atherosclerotic lesions in atherogenic ApoE −/− mice. Conclusions ENDOV is upregulated in human atherosclerotic lesions, and data from mice suggest that ENDOV promotes atherogenesis by enhancing the monocyte recruitment into the atherosclerotic lesion, potentially by increasing the effect of CCL2 activation on these cells.

Negative Correlation Between Serum Levels of Homocysteine and Apolipoprotein M

2019 ◽  
Vol 19 (2) ◽  
pp. 120-126
Author(s):  
J. Wei ◽  
Y. Yu ◽  
Y. Feng ◽  
J. Zhang ◽  
Q. Jiang ◽  
...  
Keyword(s):  
Negative Correlation ◽  
Hepg2 Cells ◽  
Serum Levels ◽  
Significant Negative Correlation ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Apolipoprotein M ◽  
Protein Mass ◽  
Protein Levels ◽  
Phosphoinositide 3 Kinase

Background: Homocysteine (Hcy) has been suggested as an independent risk factor for atherosclerosis. Apolipoprotein M (apoM) is a constituent of the HDL particles. The goal of this study was to examine the serum levels of homocysteine and apoM and to determine whether homocysteine influences apoM synthesis. Methods: Serum levels of apoM and Hcy in 17 hyperhomocysteinemia (HHcy) patients and 19 controls were measured and their correlations were analyzed. Different concentrations of homocysteine (Hcy) and LY294002, a specific phosphoinositide 3- kinase (PI3K) inhibitor, were used to treat HepG2 cells. The mRNA levels were determined by RT-PCR and the apoM protein mass was measured by western blot. Results: We found that decreased serum apoM levels corresponded with serum HDL levels in HHcy patients, while the serum apoM levels showed a statistically significant negative correlation with the serum Hcy levels. Moreover, apoM mRNA and protein levels were significantly decreased after the administration of Hcy in HepG2 cells, and this effect could be abolished by addition of LY294002. Conclusions: resent study demonstrates that Hcy downregulates the expression of apoM by mechanisms involving the PI3K signal pathway.

scholarly journals OCT angiography metrics predict intradialytic hypotension episodes in chronic hemodialysis patients: a pilot, prospective study

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Giuseppe Coppolino ◽  
Adriano Carnevali ◽  
Valentina Gatti ◽  
Caterina Battaglia ◽  
Giorgio Randazzo ◽  
...  
Keyword(s):  
Chronic Hemodialysis ◽  
Dialysis Session ◽  
Intradialytic Hypotension ◽  
Inverse Association ◽  
Non Invasive ◽  
Kaplan Meier ◽  
Optimal Thresholds ◽  
Scanning Area ◽  
Before And After ◽  
Capillary Plexus

AbstractIn chronic hemodialysis (HD) patients, intradialytic hypotension (IDH) is a complication that increases mortality risk. We run a pilot study to analyzing possible relationships between optical coherence tomography angiography (OCT-A) metrics and IDH with the aim of evaluating if OCT-A could represent a useful tool to stratify the hypotensive risk in dialysis patients. A total of 35 eyes (35 patients) were analyzed. OCT-A was performed before and after a single dialysis session. We performed OCT-A 3 × 3 mm and 6 × 6 mm scanning area focused on the fovea centralis. Patients were then followed up to 30 days (10 HD sessions) and a total of 73 IDHs were recorded, with 12 patients (60%) experiencing at least one IDH. Different OCT-A parameters were reduced after dialysis: central choroid thickness (CCT), 6 × 6 mm foveal whole vessel density (VD) of superficial capillary plexus (SPC) and 6 × 6 mm foveal VD of deep capillary plexus (DCP). At logistic regression analysis, IDH was positively associated with baseline foveal VD of SCP and DCP, while an inverse association was found with the choroid. In Kaplan–Meier analyses of patients categorized according to the ROC-derived optimal thresholds, CCT, the 3 × 3 foveal VD of SCP, the 3 × 3 mm and 6 × 6 mm foveal VD of DCP and the 6 × 6 mm foveal VD of SCP were strongly associated with a higher risk of IDH over the 30-days follow-up. In HD patients, a single OCT-A measurement may represent a non-invasive, rapid tool to evaluate the compliance of vascular bed to HD stress and to stratify the risk of IDH in the short term.

Placement, Performance and Complications of the Ash Split Cath™ Hemodialysis Catheter

2002 ◽  
Vol 25 (12) ◽  
pp. 1137-1143 ◽  
Author(s):  
M. Gallieni ◽  
P.A. Conz ◽  
E. Rizzioli ◽  
A. Butti ◽  
D. Brancaccio
Keyword(s):  
Vascular Access ◽  
Good Alternative ◽  
Chronic Hemodialysis ◽  
Dialysis Session ◽  
Dialysis Dose ◽  
Hemodialysis Catheter ◽  
Venous Fistula ◽  
Tunneled Catheter ◽  
Urea Reduction Ratio

A tunneled catheter is the alternative vascular access for those patients in need of hemodialysis who cannot undergo dialysis through an arterio-venous fistula or a vascular graft. This study was undertaken to evaluate the performance of the Ash Split Cath™, a 14 French chronic hemodialysis catheter with D-shaped lumens and a Dacron® cuff. After tunneling through a transcutaneous portion the catheter enters the venous system, where it splits into two separate limbs. Data regarding catheter positioning, function and adequacy of dialysis were collected from two hemodialysis facilities. Twenty-eight Ash-split catheters were placed in 28 patients, with no complications, and immediate technical success was 100%. Patients were followed up for a total of 7,286 catheter days. No catheter-related infections were observed. Only one catheter failed after 15 days, with a primary catheter patency of 96% for the whole study length. Mean blood flow was 303 ± 20 ml/min at 1 week after insertion, 306 ± 17 ml/min at 3 months, 299 ± 44 ml/min at 6 months, and 308 ± 16 ml/min at 12 months. With a mean dialysis session duration of 234 ± 25 minutes, adequate dialysis dose was observed for 96% of catheters, as reflected by a mean urea reduction ratio (URR) of 71%±8 or a mean urea kinetic modeling, or Kt/V, value of 1.51±0.3 during follow up. In conclusion, compared with previous studies we report the best permanent catheter performance, confirming that the Ash-split catheter is a good alternative for vascular access in hemodialysis patients who are not candidates for surgical A-V fistula or graft placement.

Abstract 1839: MicroRNA-499 Promotes the Differentiation of Cardiac Progenitor Cells into Myocytes

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Toru Hosoda ◽  
Konrad Urbanek ◽  
Adriana Bastos Carvalho ◽  
Claudia Bearzi ◽  
Silvana Bardelli ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Progenitor Cells ◽  
Target Genes ◽  
Brdu Incorporation ◽  
Cardiac Progenitor Cells ◽  
Partial Recovery ◽  
Rt Pcr ◽  
Protein Levels ◽  
Cell Proliferation And Differentiation ◽  
Reporter Assays

Myocardial regeneration mediated by cardiac progenitor cells (CPCs) results in the partial recovery of the infarcted heart but the newly formed myocytes within the necrotic tissue have fetal-neonatal characteristics. In contrast, CPC activation in the remote viable myocardium results in the formation of mature myocytes, suggesting that CPC differentiation is conditioned by the surrounding cells. Thus, the hypothesis is raised that microRNAs (miRs) that are highly expressed in myocytes and are absent in CPCs, may translocate through gap junctions to adjacent CPCs promoting their differentiation. By employing miR array and Q-RT-PCR, miR-499 was found to be ~500-fold more expressed in myocytes than CPCs. Additionally, we demonstrated that miR-499 translocates from neighboring cells to CPCs through the formation of gap junctions. The translocated miR-499 was functional and repressed the expression of target genes. Among 200 putative targets of miR-499, we have elected to study Sox6 and Rod1. The validation of these putative miR-499-targets was obtained by reporter assays; cells transfected with miR-499 together with plasmids carrying luciferase and the 3′-UTR region of Sox6 or Rod1 show the expected decrease in luciferase activity. Transcripts of Sox6 and Rod1 were measured by Q-RT-PCR in myocytes and CPCs; Sox6 mRNA was 2-fold higher and Rod1 mRNA was 98% lower in myocytes than CPCs. However, the protein levels of Sox6 and Rod1 were significantly lower in myocytes than CPCs suggesting that miR-499 promotes degradation and/or inhibition of translation of these target genes. To document miR-499 function, CPCs were transfected with a miR-499-expression vector and cell proliferation and differentiation were evaluated 3 days later. BrdU incorporation decreased 60% and the cells displayed a marked upregulation of the myocyte-specific transcription factors Nkx2.5 and MEF2C. Similar results were obtained when Sox6 and Rod1 were selectively blocked with siRNA. In both cases, the number of Nkx2.5- and MEF2C-positive cells increased 2–3-fold. Thus, our data indicate that miR-499 translocates via gap junction from myocytes to CPCs where miR-499 is a crucial modulator of the differentiation of CPCs into cardiomyocytes through the repression of Sox6 and Rod1.

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