Myocardial maladaptation to pressure overload in CB2 receptor-deficient mice.

BACKGROUND: Adaptation to aortic valve stenosis leads to myocardial hypertrophy, which has been associated with inflammation, fibrosis and activation of the endocannabinoid system. Since the endocannabinoid system and the CB2 receptor provide cardioprotection and modulate immune response in experimental ischemia, we investigated the role of CB2 in a mouse model of cardiac pressure overload. METHODS: Transverse aortic constriction was performed in CB2 receptor-deficient (Cnr2-/-) mice and their wild-type littermates (Cnr2+/+). After echocardiography and Millar left heart catheter hemodynamic evaluation hearts were processed for histological, cellular and molecular analyses. RESULTS: The endocannabinoid system showed significantly higher anandamide production and CB2 receptor expression in Cnr2+/+ mice. Histology showed non-confluent, interstitial fibrosis with rare small areas of cardiomyocyte loss in Cnr2+/+ mice. In contrast, extensive cardiomyocyte loss and confluent scar formation were found in Cnr2-/- mice accompanied by significantly increased apoptosis and left ventricular dysfunction when compared with Cnr2+/+ mice. The underlying cardiac maladaptation in Cnr2-/- mice was associated with significantly reduced expression of myosin heavy chain isoform beta and less production of heme oxygenase-1. Cnr2-/- hearts presented after 7 days with stronger proinflammatory response including significantly higher TNF-alpha expression and macrophage density, but lower density of CD4+ and B220+ cells. At the same time, we found increased apoptosis of macrophages and adaptive immune cells. Higher myofibroblast accumulation and imbalance in MMP/TIMP-regulation indicated adverse remodeling in Cnr2-/- mice. CONCLUSIONS: Our study provides mechanistic evidence for the role of the endocannabinoid system in myocardial adaptation to pressure overload in mice. The underlying mechanisms include production of anandamide, adaptation of contractile elements and antioxidative enzymes, and selective modulation of immune cells action and apoptosis in order to prevent the loss of cardiomyocytes.

The endocannabinoid-CB2 receptor axis protects the ischemic heart at the early stage of cardiomyopathy.

Ischemic heart disease is associated with inflammation, interstitial fibrosis and ventricular dysfunction prior to the development of heart failure. Endocannabinoids and the cannabinoid receptor CB2 have been claimed to be involved, but their potential role in cardioprotection is not well understood. We therefore explored the role of the cannabinoid receptor CB2 during the initial phase of ischemic cardiomyopathy development prior to the onset of ventricular dysfunction or infarction. Wild type and CB2-deficient mice underwent daily brief, repetitive ischemia and reperfusion (I/R) episodes leading to ischemic cardiomyopathy. The relevance of the endocannabinoid-CB2 receptor axis was underscored by the finding that CB2 was upregulated in ischemic wild type cardiomyocytes and that anandamide level was transiently increased during I/R. CB2-deficient mice showed an increased rate of apoptosis, irreversible loss of cardiomyocytes and persistent left ventricular dysfunction 60 days after the injury, whereas wild type mice presented neither morphological nor functional defects. These defects were due to lack of cardiomyocyte protection mechanisms, as CB2-deficient hearts were in contrast to controls unable to induce switch in myosin heavy chain isoforms, antioxidative enzymes and chemokine CCL2 during repetitive I/R. In addition, a prolonged inflammatory response and adverse myocardial remodeling were found in CB2-deficient hearts because of postponed activation of the M2a macrophage subpopulation. Therefore, the endocannabinoid-CB2 receptor axis plays a key role in cardioprotection during the initial phase of ischemic cardiomyopathy development.

Combined use of magnetic microbeads for endothelial cell isolation and enhanced cell engraftment in myocardial repair.

Background: The regenerative potential of the heart after injury is limited. Therefore, cell replacement strategies have been developed. However, the engraftment of transplanted cells in the myocardium is very inefficient. In addition, the use of heterogeneous cell populations precludes the reproducibility of the outcome. Methods: To address both issues, in this proof of principle study, we applied magnetic microbeads for combined isolation of eGFP+ embryonic cardiac endothelial cells (CECs) by antigen-specific magnet-associated cell sorting (MACS) and improved engraftment of these cells in myocardial infarction by magnetic fields. Results: MACS provided CECs of high purity decorated with magnetic microbeads. In vitro experiments revealed that the angiogenic potential of microbead-labeled CECs was preserved and the magnetic moment of the cells was strong enough for site-specific positioning by a magnetic field. After myocardial infarction in mice, intramyocardial CEC injection in the presence of a magnet resulted in a strong improvement of cell engraftment and eGFP+ vascular network formation in the hearts. Hemodynamic and morphometric analysis demonstrated augmented heart function and reduced infarct size only when a magnetic field was applied. Conclusion: Thus, the combined use of magnetic microbeads for cell isolation and enhanced cell engraftment in the presence of a magnetic field is a powerful approach to improve cell transplantation strategies in the heart.

Live monitoring of small vessels during development and disease using the flt-1 promoter element.

Vessel formation is of critical importance for organ function in the normal and diseased state. In particular, the labeling and quantitation of small vessels prove to be technically challenging using current approaches. We have, therefore, established a transgenic embryonic stem (ES) cell line and a transgenic mouse model where the vascular endothelial growth factor receptor VEGFR-1 (flt-1) promoter drives the expression of the live reporter eGFP. Fluorescence microscopy and immunostainings revealed endothelial-specific eGFP labeling of vascular networks. The expression pattern recapitulates that of the endogenous flt-1 gene, because small and large vessels are labeled by eGFP during embryonic development; after birth, the expression becomes more restricted to small vessels. We have explored this in the cardiovascular system more in detail and found that all small vessels and capillaries within the heart are strongly eGFP+. In addition, myocardial injuries have been induced in transgenic mice and prominent vascular remodeling, and an increase in endothelial cell area within the peri-infarct area could be observed underscoring the utility of this mouse model. Thus, the transgenic flt-1/eGFP models are powerful tools to investigate and quantify vascularization in vivo and to probe the effect of different compounds on vessel formation in vitro.

Activation of Endocannabinoid System Is Associated with Persistent Inflammation in Human Aortic Aneurysm.

Human aortic aneurysms have been associated with inflammation and vascular remodeling. Since the endocannabinoid system modulates inflammation and tissue remodeling, we investigated its components in human aortic aneurysms. We obtained anterior aortic wall samples from patients undergoing elective surgery for aortic aneurysm or coronary artery disease as controls. Histological and molecular analysis (RT-qPCR) was performed, and endocannabinoid concentration was determined using LC-MRM. Patient characteristics were comparable between the groups except for a higher incidence of arterial hypertension and diabetes in the control group. mRNA level of cannabinoid receptors was significantly higher in aneurysms than in controls. Concentration of the endocannabinoid 2-arachidonoylglycerol was significantly higher, while the second endocannabinoid anandamide and its metabolite arachidonic acid and palmitoylethanolamide were significantly lower in aneurysms. Histology revealed persistent infiltration of newly recruited leukocytes and significantly higher mononuclear cell density in adventitia of the aneurysms. Proinflammatory environment in aneurysms was shown by significant upregulation of M-CSF and PPARγ but associated with downregulation of chemokines. We found comparable collagen-stained area between the groups, significantly decreased mRNA level of CTGF, osteopontin-1, and MMP-2, and increased TIMP-4 expression in aneurysms. Our data provides evidence for endocannabinoid system activation in human aortic aneurysms, associated with persistent low-level inflammation and vascular remodeling.

CB2 receptor-mediated effects of pro-inflammatory macrophages influence survival of cardiomyocytes.

AIMS: The endocannabinoid system and cannabinoid receptor 2 (CB2 receptor) have been associated with modulation of inflammatory response and myocardial adaptation after ischemic injury. In order to elucidate CB2 receptor-related effects during cellular interactions, we investigated cardiomyocyte survival and macrophage function in vitro. MAIN METHODS: Murine embryonic (eCM) and adult (CM) cardiomyocytes, murine macrophages (MO), and their subtypes M1 (M1-MO) and M2 (M2-MO) were derived from wildtype- (WT) and CB2 receptor-deficient (Cnr2(-/-)) mice. Cells were cultured separately or in co-culture under normoxia or hypoxia (2% O2) and pro-inflammatory stimulation using interferon (IFN)γ. Besides immunohistochemistry, we also measured mRNA expression (Taqman®) and performed FACS-analysis of cardiomyocytes. Macrophage migration was assessed using Boyden chamber assay. KEY FINDINGS: We found a significant induction of CB2 receptor mRNA and protein in murine eCM as well as M1- and M2-MO in vitro following cultivation under hypoxia or stimulation with IFNγ. A significantly higher amount of apoptotic Cnr2(-/-)-CMs was found after incubation under hypoxia when compared to WT-CMs. We observed a significantly stronger migration potential in Cnr2(-/-)-M1-MOs towards the supernatant of apoptotic CM, than in corresponding WT-cells. Co-culture revealed a significantly higher loss of eCMs and induction of their apoptosis after cultivation with Cnr2(-/-)-M1-MOs. Production of TNF-α in M1-MOs was dependent on CB2 receptor stimulation by anandamide. SIGNIFICANCE: Our data provide novel insights into CB2 receptor-mediated protection of cardiomyocytes during hypoxia and pro-inflammatory stimulation. We show CB2 receptor-dependent effects on migration and function of M1-MOs in interaction with cardiomyocytes, thereby influencing their survival.

Impaired border zone formation and adverse remodeling after reperfused myocardial infarction in cannabinoid CB2 receptor deficient mice.

AIMS: Reperfusion ofmyocardial infarction is associated with inflammatory reaction and subsequentmyocardial remodeling with a rapid scar formation in mice. The cannabinoid receptor CB2 has been associated with cardioprotection and regulation ofmacrophage function.Weinvestigated its role in remodeling of reperfused infarction. MAIN METHODS: One hour LAD-occlusion was followed by reperfusion over 6 h and 1, 3 and 7 days in wild-type C57/BL6J (WT) and CB2 receptor-deficient (Cnr2−/−)mice (n=8/group). Hearts were processed for functional, morphological and mRNA/protein analysis, and tissue concentration of endocannabinoidswas determined using liquid chromatography-multiple reaction monitoring. KEY FINDINGS: In contrast to a rapid formation of granulation tissue and a compacted non-transmural scar inWT mice after 7 days of reperfusion, Cnr2−/− mice showed a non-compacted transmural scar. Millar® left ventricular catheter measurements revealed a significantly worse function in Cnr2−/− mice.We found no compensatory elevation of endocannabinoid concentration in Cnr2−/− hearts. Macrophage infiltration was significantly stronger in Cnr2−/− hearts and affected also the remote septum, when compared to WT hearts.We found a cytokine-driven inflammatory response in Cnr2−/− hearts with no significant induction of chemokines. Immunohistochemistry for thrombospondin-1 revealed a dysfunctional infarction border zone formation in Cnr2−/− hearts. Cnr2−/−hearts showed no significant induction of tenascin C, collagen-Iα or lysil oxidase, thereby indicating adversemyocardial remodeling. SIGNIFICANCE: Endocannabinoids act via CB2 receptor in the modulation of inflammatory response and myocardial remodeling after infarction. CB2 receptor plays an important role in the formation of infarction border zone, collagen deposition and organization of stable scar during remodeling.

Cardiomyocyte specific peroxisome proliferator-activated receptor-α overexpression leads to irreversible damage in ischemic murine heart.

AIMS: Peroxisome proliferator-activated receptor (PPAR)-α is downregulated in ischemic myocardium resulting in substrate switch from fatty acid oxidation to glucose utilization. Pharmacological PPAR-α activation leads to increased fatty acid oxidation and myocardial lipotoxicity. The aim of our study was to investigate the role of cardiomyocyte specific PPAR-α overexpression in myocardial adaptation to repetitive ischemic injury without myocardial infarction. MAIN METHODS: Repetitive, brief I/R was performed in male and female MHC-PPAR-α overexpressing and wildtype-C57/Bl6 (WT)-mice, age 10-12 weeks, for 3 and 7 consecutive days. After echocardiography, their hearts were excised for histology and gene/protein-expression measurements (Taqman/Western blot). KEY FINDINGS: MHC-PPAR-α mice developed microinfarctions already after 3 days of repetitive I/R in contrast to interstitial fibrosis in WT-mice. We found higher deposition of glycogen, increased apoptosis and dysfunctional regulation of antioxidative mediators in MHC-PPAR-α mice. MHC-PPAR-α mice presented with maladaptation of myosin heavy chain isoforms and worse left ventricular dysfunction than WT-mice. We found prolonged, chemokine-driven macrophage infiltration without induction of proinflammatory cytokines in MHC-PPAR-α mice. Persistent accumulation of myofibroblasts in microinfarctions indicated active remodeling resulting in scar formation in contrast to interstitial fibrosis without microinfarctions in WT-mice. However, MHC-PPAR-α hearts had only a weak induction of tenascin-C in contrast to its strong expression in WT-hearts. SIGNIFICANCE: Cardiomyocyte-specific PPAR-α overexpression led to irreversible cardiomyocyte loss with deteriorated ventricular function during brief, repetitive I/R episodes. We identified higher glycogen deposition, increased apoptosis, deranged antioxidative capacity and maladaptation of contractile elements as major contributors involved in the modulation of post-ischemic inflammation and remodeling.

Cardioprotective effects of osteopontin-1 during development of murine ischemic cardiomyopathy.

Repetitive brief ischemia and reperfusion (I/R) is associated with ventricular dysfunction in pathogenesis of murine ischemic cardiomyopathy and human hibernating myocardium. We investigated the role of matricellular protein osteopontin-1 (OPN) in murine model of repetitive I/R. One 15-min LAD-occlusion followed by reperfusion was performed daily over 3, 5, and 7 consecutive days in C57/Bl6 wildtype- (WT-) and OPN(-/-)-mice (n = 8/group). After echocardiography hearts were processed for histological and mRNA-studies. Cardiac fibroblasts were isolated, cultured, and stimulated with TGF- ß 1. WT-mice showed an early, strong, and cardiomyocyte-specific osteopontin-expression leading to interstitial macrophage infiltration and consecutive fibrosis after 7 days I/R in absence of myocardial infarction. In contrast, OPN(-/-)-mice showed small, nontransmural infarctions after 3 days I/R associated with significantly worse ventricular dysfunction. OPN(-/-)-mice had different expression of myocardial contractile elements and antioxidative mediators and a lower expression of chemokines during I/R. OPN(-/-)-mice showed predominant collagen deposition in macrophage-rich small infarctions. We found lower induction of tenascin-C, MMP-9, MMP-12, and TIMP-1, whereas MMP-13-expression was higher in OPN(-/-)-mice. Cultured OPN(-/-)-myofibroblasts confirmed these findings. In conclusion, osteopontin seems to modulate expression of contractile elements, antioxidative mediators, and inflammatory response and subsequently remodel in order to protect cardiomyocytes in murine ischemic cardiomyopathy.

Myocardial hypertrophy is associated with inflammation and activation of endocannabinoid system in patients with aortic valve stenosis.

AIMS: Endocannabinoids and their receptors have been associated with cardiac adaptation to injury, inflammation and fibrosis. Experimental studies suggested a role for inflammatory reaction and active remodeling in myocardial hypertrophy, but they have not been shown in human hypertrophy. We investigated the association of the endocannabinoid system with myocardial hypertrophy in patients with aortic stenosis. MAIN METHODS: Myocardial biopsies were collected from patients with aortic stenosis (AS) and atrial myxoma as controls during surgery. Histological and molecular analysis of endocannabinoids and their receptors, inflammatory and remodeling-related cells and mediators was performed. KEY FINDINGS: Myocardial hypertrophy was confirmed with significantly higher cardiomyocyte diameter in AS than in myxoma patients, which had normal cell size. AS patients presented compensated myocardial adaptation to pressure overload. AS patients had significantly higher: concentration of endocannabinoid anandamide, expression of its degrading enzyme FAAH, and of cannabinoid receptor CB2, being predominantly located on cardiomyocytes. Cell density of macrophages and newly recruited leukocytes were higher in AS group, which together with increased expression of chemokines CCL2, CCL4 and CXCL8, and suppression of anti-inflammatory IL-10 indicates persistent inflammatory reaction. We found higher myofibroblast density and stronger tenascin C staining along with mRNA induction of tenascin C and CTGF in AS patients showing active myocardial remodeling. SIGNIFICANCE: Our study shows for the first time activation of the endocannabinoid system and predominant expression of its receptor CB2 on cardiomyocytes being associated with persistent inflammation and active remodeling in hypertrophic myocardium of patients with aortic stenosis.

Identification of a novel vasoconstrictor peptide specific for the systemic circulation.

Some small molecular weight peptides possess potent vasoactive properties. Herein we have identified the laminin nonapeptide (LNP) CDPGYIGSR as a novel vasoconstrictive agent. Isometric force measurements revealed that LNP induced vasoconstriction in small and large murine arteries in a dose-dependent fashion; LNP also increased vascular tone in human mammary arteries. The vasoactive response was specific for the nonapeptide, because neither scrambled nor very similar peptide sequences modulated vascular tone. As an underlying mechanism we found in [Ca(2+)](i) imaging experiments that the nonapeptide induced transmembrane [Ca(2+)](i) influx in vascular smooth muscle cells. Patch clamp experiments showed that LNP activated nonselective cation channels, causing depolarization of the membrane potential and opening of L-type Ca(2+) channels. The functional effect of LNP was also assessed with catheter measurements in mice in vivo and confirmed vasoconstriction. This effect was restricted to the systemic circulation, because measurements with the perfused lung system demonstrated that LNP did not alter vascular tone in pulmonary arteries. Thus, LNP is a vasoconstrictor in mouse and human arteries, and its vasoactivity is restricted to the systemic vasculature.