Hector A. Cabrera-Fuentes

Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice

Background— Atherosclerosis and vascular remodeling after injury are driven by inflammation and mononuclear cell infiltration. Extracellular RNA (eRNA) has recently been implicated to become enriched at sites of tissue damage and to act as a proinflammatory mediator. Here, we addressed the role of eRNA in high-fat diet–induced atherosclerosis and neointima formation after injury in atherosclerosis-prone mice. Methods and Results— The presence of eRNA was revealed in atherosclerotic lesions from high-fat diet–fed low-density lipoprotein receptor–deficient (Ldlr−/−) mice in a time-progressive fashion. RNase activity in plasma increased within the first 2 weeks (44±9 versus 70±7 mU/mg protein; P=0.0012), followed by a decrease to levels below baseline after 4 weeks of high-fat diet (44±9 versus 12±2 mU/mg protein; P<0.0001). Exposure of bone marrow–derived macrophages to eRNA resulted in a concentration-dependent upregulation of the proinflammatory mediators tumor necrosis factor-&agr;, arginase-2, interleukin-1&bgr;, interleukin-6, and interferon-&ggr;. In a model of accelerated atherosclerosis after arterial injury in apolipoprotein E–deficient (ApoE−/−) mice, treatment with RNase1 diminished the increased plasma level of eRNA evidenced after injury. Likewise, RNase1 administration reduced neointima formation in comparison with vehicle-treated ApoE−/− controls (25.0±6.2 versus 46.9±6.9×103 &mgr;m2, P=0.0339) and was associated with a significant decrease in plaque macrophage content. Functionally, RNase1 treatment impaired monocyte arrest on activated smooth muscle cells under flow conditions in vitro and inhibited leukocyte recruitment to injured carotid arteries in vivo. Conclusions— Because eRNA is associated with atherosclerotic lesions and contributes to inflammation-dependent plaque progression in atherosclerosis-prone mice, its targeting with RNase1 may serve as a new treatment option against atherosclerosis.

Mitochondrial fusion and fission proteins as novel therapeutic targets for treating cardiovascular disease.

The past decade has witnessed a number of exciting developments in the field of mitochondrial dynamics – a phenomenon in which changes in mitochondrial shape and movement impact on cellular physiology and pathology. By undergoing fusion and fission, mitochondria are able to change their morphology between elongated interconnected networks and discrete fragmented structures, respectively. The cardiac mitochondria, in particular, have garnered much interest due to their unique spatial arrangement in the adult cardiomyocyte, and the multiple roles they play in cell death and survival. In this article, we review the role of the mitochondrial fusion and fission proteins as novel therapeutic targets for treating cardiovascular disease.

Role of the MPTP in conditioning the heart – translatability and mechanism

Mitochondria have long been known to be the gatekeepers of cell fate. This is particularly so in the response to acute ischaemia‐reperfusion injury (IRI). Following an acute episode of sustained myocardial ischaemia, the opening of the mitochondrial permeability transition pore (MPTP) in the first few minutes of reperfusion, mediates cell death. Preventing MPTP opening at the onset of reperfusion using either pharmacological inhibitors [such as cyclosporin A (CsA) ] or genetic ablation has been reported to reduce myocardial infarct (MI) size in animal models of acute IRI. Interestingly, the endogenous cardioprotective intervention of ischaemic conditioning, in which the heart is protected against MI by applying cycles of brief ischaemia and reperfusion to either the heart itself or a remote organ or tissue, appears to be mediated through the inhibition of MPTP opening at reperfusion. Small proof‐of‐concept clinical studies have demonstrated the translatability of this therapeutic approach to target MPTP opening using CsA in clinical settings of acute myocardial IRI. However, given that CsA is a not a specific MPTP inhibitor, more novel and specific inhibitors of the MPTP need to be discovered – the molecular identification of the MPTP should facilitate this. In this paper, we review the role of the MPTP as a target for cardioprotection, the potential mechanisms underlying MPTP inhibition in the setting of ischaemic conditioning, and the translatability of MPTP inhibition as a therapeutic approach in the clinical setting.

Impact of extracellular RNA on endothelial barrier function

Different types of high and low molecular weight extracellular RNA (eRNA) are liberated from cells upon conditions of tissue damage or vascular diseases and have been demonstrated in vivo and in vitro to influence the integrity and barrier function of the vascular endothelium. Among the types of self eRNA studied in this respect, ribosomal RNA appears to engage cytokines to promote hyperpermeability, while counteracting RNase1 serves as a potent vessel-protective factor. Different microRNAs may change the expression program of endothelial cells with consequences for cellular contacts and stability. Non-self viral RNAs are recognized by Toll-like receptors that transmit intracellular inflammation signals to disturb the vascular barrier function, largely in connection with infectious diseases. Although derived from the same nucleotide building blocks, the various forms of eRNA exhibit a multitude of molecular interactions with the endothelium that may drastically change its phenotypical characteristics. The impact of eRNA on vascular integrity in health and disease is summarized in this concise review.

Hmga2 is required for canonical WNT signaling during lung development

BackgroundThe high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. HMG proteins are present at high levels in various undifferentiated tissues during embryonic development and their levels are strongly reduced in the corresponding adult tissues, where they have been implicated in maintaining and activating stem/progenitor cells. Here we deciphered the role of the high-mobility-group AT-hook protein 2 (HMGA2) during lung development by analyzing the lung of Hmga2-deficient mice (Hmga2−/−).ResultsWe found that Hmga2 is expressed in the mouse embryonic lung at the distal airways. Analysis of Hmga2−/− mice showed that Hmga2 is required for proper cell proliferation and distal epithelium differentiation during embryonic lung development. Hmga2 knockout led to enhanced canonical WNT signaling due to an increased expression of secreted WNT glycoproteins Wnt2b, Wnt7b and Wnt11 as well as a reduction of the WNT signaling antagonizing proteins GATA-binding protein 6 and frizzled homolog 2. Analysis of siRNA-mediated loss-of-function experiments in embryonic lung explant culture confirmed the role of Hmga2 as a key regulator of distal lung epithelium differentiation and supported the causal involvement of enhanced canonical WNT signaling in mediating the effect of Hmga2-loss-of-fuction. Finally, we found that HMGA2 directly regulates Gata6 and thereby modulates Fzd2 expression.ConclusionsOur results support that Hmga2 regulates canonical WNT signaling at different points of the pathway. Increased expression of the secreted WNT glycoproteins might explain a paracrine effect by which Hmga2-knockout enhanced cell proliferation in the mesenchyme of the developing lung. In addition, HMGA2-mediated direct regulation of Gata6 is crucial for fine-tuning the activity of WNT signaling in the airway epithelium. Our results are the starting point for future studies investigating the relevance of Hmga2-mediated regulation of WNT signaling in the adult lung within the context of proper balance between differentiation and self-renewal of lung stem/progenitor cells during lung regeneration in both homeostatic turnover and repair after injury.

From basic mechanisms to clinical applications in heart protection, new players in cardiovascular diseases and cardiac theranostics: meeting report from the third international symposium on "New frontiers in cardiovascular research"

In this meeting report, particularly addressing the topic of protection of the cardiovascular system from ischemia/reperfusion injury, highlights are presented that relate to conditioning strategies of the heart with respect to molecular mechanisms and outcome in patients’ cohorts, the influence of co-morbidities and medications, as well as the contribution of innate immune reactions in cardioprotection. Moreover, developmental or systems biology approaches bear great potential in systematically uncovering unexpected components involved in ischemia–reperfusion injury or heart regeneration. Based on the characterization of particular platelet integrins, mitochondrial redox-linked proteins, or lipid-diol compounds in cardiovascular diseases, their targeting by newly developed theranostics and technologies opens new avenues for diagnosis and therapy of myocardial infarction to improve the patients’ outcome.

Meeting report from the 2nd International Symposium on New Frontiers in Cardiovascular Research. Protecting the cardiovascular system from ischemia: between bench and bedside

Recent advances in basic cardiovascular research as well as their translation into the clinical situation were the focus at the last “New Frontiers in Cardiovascular Research meeting”. Major topics included the characterization of new targets and procedures in cardioprotection, deciphering new players and inflammatory mechanisms in ischemic heart disease as well as uncovering microRNAs and other biomarkers as versatile and possibly causal factors in cardiovascular pathogenesis. Although a number of pathological situations such as ischemia–reperfusion injury or atherosclerosis can be simulated and manipulated in diverse animal models, also to challenge new drugs for intervention, patient studies are the ultimate litmus test to obtain unequivocal information about the validity of biomedical concepts and their application in the clinics. Thus, the open and bidirectional exchange between bench and bedside is crucial to advance the field of ischemic heart disease with a particular emphasis of understanding long-lasting approaches in cardioprotection.

RNase1 as a potential mediator of remote ischaemic preconditioning for cardioprotection

OBJECTIVES Remote ischaemic preconditioning (RIPC) is a non-invasive and virtually cost-free strategy for protecting the heart against acute ischaemia-reperfusion injury (IRI). We have recently shown that the inhibition of extracellular RNA (eRNA) using non-toxic RNase1 protected the heart against acute IRI, reduced myocardial infarct (MI) size and preserved left ventricular systolic function in rodent animal MI models. Based on this previous work in animals, the role of the eRNA/RNase1 system in cardiac RIPC in humans should be defined. METHODS Fourteen patients underwent cardiac surgery without RIPC; from each patient, six separate 5 ml blood specimens from radial artery and two blood specimens from coronary sinus at different time points during heart surgery were taken. Six healthy donors received RIPC (4 × 5 min upper limb ischaemia); blood parameters were quantified before and after RIPC. Twelve patients underwent cardiac surgery of which 6 received RIPC, whereas the remaining 6 were exposed to sham procedure. Circulating eRNA was quantified in plasma from arterial and coronary sinus blood obtained from patients undergoing cardiac by standard procedures. Tumour necrosis factor-α (TNF-α) production by heart tissue was assessed by enzyme-linked immuno-sorbent assay; RNase activity was quantified by an enzymatic assay. RESULTS Before surgery, eRNA levels were similar in both groups (14 ± 6 vs 13 ± 5 ng/ml; P = 0.9967). In patients without RIPC, arterial eRNA levels rose during surgery (87 ± 12 ng/ml) and peaked after (127 ± 11 ng/ml) aortic declamping; accordingly, eRNA levels in coronary sinus blood were significantly higher (206 ± 32 ng/ml; P = 0.0129) than that in radial artery. Moreover, significant elevation of TNF-α (36 ± 6 ng/ml; P = 0.0059) particularly in coronary sinus blood after opening of the aortic clamping was observed. Interestingly, applying a RIPC protocol significantly increased levels of plasma endogenous vascular RNase1 by >7-fold, and the levels of arterial (31 ± 7 ng/ml; P = 0.0024) and coronary sinus (37 ± 9 ng/ml; P < 0.0001) circulating eRNA, as well as circulating TNF-α (20 ± 4 ng/ml; P = 0.0050) levels were significantly reduced. CONCLUSIONS Upon RIPC, the level of cardioprotective RNase1 increased, while the concentration of damaging eRNA and TNF-α decreased. The present findings imply a significant contribution of the RIPC-dependent (endothelial) RNase1 for improving the outcome of cardiac surgery. However, the exact mechanism of RNase1-induced cardioprotection still remains to be explored.

Response to Letter Regarding Article “Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice”

We acknowledge the interest and comments of Dr Chen et al regarding our findings that self-extracellular RNA (eRNA) significantly contributes to atherogenesis (as demonstrated in 2 established animal models) by inducing a prominent inflammatory response in situ and in bone marrow–derived macrophages (BMDM), as well.1 In particular, Chen et al question whether eRNA-dependent effects may have been mediated by Toll-like receptor (TLR)–related signaling, because they recently reported that BMDM responses toward the RNA analogue poly(IC) were significantly dampened in TLR3-deficient cells.2 During the past decade, our laboratory has characterized a number of new functions of eRNA in inflammation and cardiovascular diseases. In the indicated study, we aimed to characterize a causal role of natural eRNA, which may also serve as a cell-injury marker, in the onset and progression of atherosclerosis. It …

Inflammation following acute myocardial infarction: Multiple players, dynamic roles, and novel therapeutic opportunities

ABSTRACT Acute myocardial infarction (AMI) and the heart failure that often follows, are major causes of death and disability worldwide. As such, new therapies are required to limit myocardial infarct (MI) size, prevent adverse left ventricular (LV) remodeling, and reduce the onset of heart failure following AMI. The inflammatory response to AMI, plays a critical role in determining MI size, and a persistent pro‐inflammatory reaction can contribute to adverse post‐MI LV remodeling, making inflammation an important therapeutic target for improving outcomes following AMI. In this article, we provide an overview of the multiple players (and their dynamic roles) involved in the complex inflammatory response to AMI and subsequent LV remodeling, and highlight future opportunities for targeting inflammation as a therapeutic strategy for limiting MI size, preventing adverse LV remodeling, and reducing heart failure in AMI patients.