Tumour-derived Extracellular Vesicle and Particle Reprogramming of Interstitial Macrophages in the Lung Pre-Metastatic Niche Enhances Vascular Permeability and Metastatic Potential.

Extracellular vesicles and particles (EVPs) are pivotal mediators of pre-metastatic niche formation and cancer progression, including induction of vascular permeability, which facilitates tumor cell extravasation and metastasis. However, the mechanisms through which EVPs exert this effect remain poorly understood. Here, we elucidate a novel mechanism by which tumor EVPs enhance endothelial cell permeability, tumor extravasation, and lung metastasis to different degrees, depending on tumor type. Strikingly, vascular leakiness is observed within 48h following tumor implantation and as early as one hour following intravenous injection of tumour-derived EVPs in naïve mice. Surprisingly, rather than acting directly on endothelial cells, EVPs first activate interstitial macrophages (IMs) leading to activation of JAK/STAT signaling and IL-6 secretion in IMs which subsequently promote endothelial permeability. Depletion of IMs significantly reduces tumour-derived EVP-dependent vascular leakiness and metastatic potential. Tumour EVPs that strongly induce vascular leakiness express high levels of ITGα5, and ITGα5 ablation impairs IM activation, cytokine secretion, and subsequently vascular permeability and metastasis. Importantly, IL-6 expression is elevated in IMs from non-involved tumor-adjacent lung tissue compared to distal lung tissue in lung cancer patients, highlight the clinical relevance of our discovery. Our findings identify a key role for IM activation as an initiating step in tumor type-specific EVP-driven vascular permeability and metastasis, offering promising targets for therapeutic intervention.

INO-8875, a highly selective A1 adenosine receptor agonist: evaluation of chronotropic, dromotropic, and hemodynamic effects in rats.

Selective pharmacological activation of the adenosine 1 receptor (A(1)R) is a promising new approach to achieve a potent block of atrioventricular (A-V)-nodal conduction without significant cardiovascular side effects. The purpose of the present study was to evaluate the cardiovascular profile of INO-8875, a highly selective A(1)R agonist, and to compare its properties with N-[3(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT-510), which has already been shown to induce negative dromotropic effects with minimal cardiovascular side effects in animals and in clinical studies. Dose-response experiments in the isolated hearts of rats were used to evaluate the functional selectivity of INO-8875 for the slowing of A-V-nodal conduction. Ventilated adult rats were used to study the effects of INO-8875, in vivo, on arterial blood pressure as well as on supraventricular electrophysiology. Ex vivo, INO-8875 (100 nM to 3 µM) progressively prolonged A-V-nodal conduction without reducing left ventricular function or coronary resistance. In vivo, INO-8875 up to a dose of 50 µg/kg did not reduce the carotid arterial blood pressure (n = 4). INO-8875 (1-50 µg/kg) and CVT-510 (20 and 50 µg/kg) both induced a dose-dependent decrease in heart rate and atrial refractoriness, as well as slowing of A-V-nodal conduction. However, compared with CVT-510, the activity of INO-8875 was more pronounced in A-V-nodal function. INO-8875 exhibited a greater duration of action, lasting up to 2.5 hours post dosing, whereas the effects of CVT-510 dissipated over 1 hour. INO-8875 demonstrates functional properties of a highly selective A(1)R agonist. INO-8875 exhibits an increased dromotropic effect and greater duration of action compared with CVT-510.

Primary Melanoma miRNA Trafficking Induces Lymphangiogenesis.

Melanoma, the deadliest cutaneous tumor, initiates within the epidermis; during progression, cells invade into the dermis and become metastatic through the lymphatic and blood system. Before melanoma cell invasion into the dermis, an increased density of dermal lymphatic vessels is observed, generated by a mechanism which is not fully understood. In this study, we show that, while at the primary epidermal stage (in situ), melanoma cells secrete extracellular vesicles termed melanosomes, which are uptaken by dermal lymphatic cells, leading to transcriptional and phenotypic pro-lymphangiogenic changes. Mechanistically, melanoma-derived melanosomes traffic mature let-7i to lymphatic endothelial cells, which mediate pro-lymphangiogenic phenotypic changes by the induction of type I IFN signaling. Furthermore, transcriptome analysis upon treatment with melanosomes or let-7i reveals the enhancement of IFI6 expression in lymphatic cells. Because melanoma cells metastasize primarily via lymphatic vessels, our data suggest that blocking lymphangiogenesis by repressing either melanosome release or type I IFN signaling will prevent melanoma progression to the deadly metastatic stage.

ZnT-1 enhances the activity and surface expression of T-type calcium channels through activation of Ras-ERK signaling.

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.

Prevascularization of cardiac patch on the omentum improves its therapeutic outcome.

The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modality for regenerating the myocardium after myocardial infarction (MI). We present here a strategy for the engineering of a cardiac patch with mature vasculature by heterotopic transplantation onto the omentum. The patch was constructed by seeding neonatal cardiac cells with a mixture of prosurvival and angiogenic factors into an alginate scaffold capable of factor binding and sustained release. After 48 h in culture, the patch was vascularized for 7 days on the omentum, then explanted and transplanted onto infarcted rat hearts, 7 days after MI induction. When evaluated 28 days later, the vascularized cardiac patch showed structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Thus, our study provides evidence that grafting prevascularized cardiac patch into infarct can improve cardiac function after MI.

INO-8875, a Highly-Selective A1 Adenosine Receptor Agonist: Evaluation of Chronotropic, Dromotropic and Hemodynamic Effects in Rats.

Publication of this article is suspended until the authors can provide full identification and verification of the chemical structure of INO-8875.

Exosome-Mediated Metastasis: Communication from a Distance.

Metastasis, a critical phase of tumor progression, remains a primary challenge in treating cancer and a major cause of cancer mortality. Cell-cell communication via extracellular vesicles (exosomes and microvesicles) between primary tumor cells and the microenvironment of distant organs is crucial for pre-metastatic niche (PMN) formation and metastasis. Here, we review work on the contribution of exosome cargo to cancer progression, the role of exosomes in PMN establishment, and the function of exosomes in organotropic metastasis. We also describe the clinical utility of exosomes.

Mutated MITF-E87R in Melanoma Enhances Tumor Progression via S100A4.

Melanoma, a melanocyte origin neoplasm, is the most lethal type of skin cancer, and incidence is increasing. Several familial and somatic mutations have been identified in the gene encoding the melanocyte lineage master regulator, MITF; however, the neoplastic mechanisms of these mutant MITF variants are mostly unknown. Here, by performing unbiased analysis of the transcriptomes in cells expressing mutant MITF, we identified calcium-binding protein S100A4 as a downstream target of MITF-E87R. By using wild-type and mutant MITF melanoma lines, we found that both endogenous wild-type and MITF-E87R variants occupy the S100A4 promoter. Remarkably, whereas wild-type MITF represses S100A4 expression, MITF-E87R activates its transcription. The opposite effects of wild-type and mutant MITF result in opposing cellular phenotypes, because MITF-E87R via S100A4 enhanced invasion and reduced adhesion in contrast to wild-type MITF activity. Finally, we found that melanoma patients with altered S100A4 expression have poor prognosis. These data show that a change in MITF transcriptional activity from repression to activation of S100A4 that results from a point mutation in MITF alters melanoma invasive ability. These data suggest new opportunities for diagnosis and treatment of metastatic melanoma.

Parkin Somatic Mutations Link Melanoma and Parkinson's Disease.

Epidemiological studies suggest a direct link between melanoma and Parkinson's disease (PD); however, the underlying molecular basis is unknown. Since mutations in Parkin are the major driver of early-onset PD and Parkin was recently reported to play a role in cancer development, we hypothesized that Parkin links melanoma and PD. By analyzing whole exome/genome sequencing of Parkin from 246 melanoma patients, we identified five non-synonymous mutations, three synonymous mutations, and one splice region variant in Parkin in 3.6% of the samples. In vitro analysis showed that wild-type Parkin plays a tumor suppressive role in melanoma development resulting in cell-cycle arrest, reduction of metabolic activity, and apoptosis. Using a mass spectrometry-based analysis, we identified potential Parkin substrates in melanoma and generated a functional protein association network. The activity of mutated Parkin was assessed by protein structure modeling and examination of Parkin E3 ligase activity. The Parkin-E28K mutation impairs Parkin ubiquitination activity and abolishes its tumor suppressive effect. Taken together, our analysis of genomic sequence and in vitro data indicate that Parkin is a potential link between melanoma and Parkinson's disease. Our findings suggest new approaches for early diagnosis and treatment against both diseases.

Melanoma miRNA trafficking controls tumour primary niche formation.

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

Speckle-tracking echocardiography elucidates the effect of pacing site on left ventricular synchronization in the normal and infarcted rat myocardium.

BACKGROUND: Right ventricular (RV) pacing generates regional disparities in electrical activation and mechanical function (ventricular dyssynchrony). In contrast, left ventricular (LV) or biventricular (BIV) pacing can improve cardiac efficiency in the setting of ventricular dyssynchrony, constituting the rationale for cardiac resynchronization therapy (CRT). Animal models of ventricular dyssynchrony and CRT currently relay on large mammals which are expensive and not readily available to most researchers. We developed a methodology for double-site epicardial pacing in conscious rats. Here, following post-operative recovery, we compared the effects of various pacing modes on LV dyssynchrony in normal rats and in rats with ischemic cardiomyopathy. METHODS: Two bipolar electrodes were implanted in rats as follows: Group A (n = 6) right atrial (RA) and RV sites; Group B (n = 7) RV and LV sites; Group C (n = 8) as in group B in combination with left coronary artery ligation. Electrodes were exteriorized through the back. Following post-operative recovery, two-dimensional transthoracic echocardiography was performed during pacing through the different electrodes. Segmental systolic circumferential strain (Ecc) was used to evaluate LV dyssynchrony. RESULTS: In normal rats, RV pacing induced marked LV dyssynchrony compared to RA pacing or sinus rhythm, as measured by the standard deviation (SD) of segmental time to peak Ecc, SD of peak Ecc, and the average delay between opposing ventricular segments. LV pacing and, to a greater extend BIV pacing diminished the LV dyssynchrony compared to RV pacing. In rats with extensive MI, the effects of LV and BIV pacing were markedly attenuated, and the response of individual animals was variable. CONCLUSIONS: Rodent cardiac pacing mimics important features seen in humans. This model may be developed as a simple new tool to study the pathophysiology of ventricular dyssynchrony and CRT.

ZnT-1 protects HL-1 cells from simulated ischemia-reperfusion through activation of Ras-ERK signaling.

Activation of ERK signaling may promote cardioprotection from ischemia-reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.

The involvement of ZnT-1, a new modulator of cardiac L-type calcium channels, in [corrected] atrial tachycardia remodeling. [corrected].

Atrial fibrillation (AF), the highest occurring cardiac arrhythmia in the Western world, is associated with substantial morbidity and increased mortality. In spite of extensive research, the cause of atrial electrical remodeling, a major factor in the self-perpetuating nature of AF, is still unknown. Downregulation of L-type Ca2+ channel (LTCC) activity is the hallmark of atrial electrical remodeling. ZnT-1 is a ubiquitous membrane protein that was recently suggested to inhibit the LTCC. We have studied and shown that ZnT-1 expression inhibits LTCC function in an oocyte expression system as well as in isolated cardiomyocytes. Our data also show that rapid electrical pacing can augment ZnT-1 expression in culture as well as in the atria of rats in vivo. Finally, in a pilot study, ZnT-1 expression was found to be augmented in the atria of AF patients. These findings position ZnT-1 as a probable missing link in the mechanism underlying atrial tachycardia remodeling.

New insights into the atrial electrophysiology of rodents using a novel modality: the miniature-bipolar hook electrode.

Studies of atrial electrophysiology (EP) in rodents are challenging, and available data are sparse. Herein, we utilized a novel type of bipolar electrode to evaluate the atrial EP of rodents through small lateral thoracotomy. In anesthetized rats and mice, we attached two bipolar electrodes to the right atrium and a third to the right ventricle. This standard setup enabled high-resolution EP studies. Moreover, a permanent implantation procedure enabled EP studies in conscious freely moving rats. Atrial EP was evaluated in anesthetized rats, anesthetized mice (ICR and C57BL6 strains), and conscious rats. Signal resolution enabled atrial effective refractory period (AERP) measurements and first time evaluation of the failed 1:1 atrial capture, which was unexpectedly longer than the AERP recorded at near normal cycle length by 27.2+/-2.3% in rats (P<0.0001; n=35), 31.7+/-8.3% in ICR mice (P=0.0001; n=13), and 57.7+/-13.7% in C57BL6 mice (P=0.015; n=4). While AERP rate adaptation was noted when 10 S1s at near normal basic cycle lengths were followed by S2 at varying basic cycle length and S3 for AERP evaluation, such rate adaptation was absent using conventional S1S2 protocols. Atrial tachypacing in rats shortened the AERP values on a timescale of hours, but a reverse remodeling phase was noted thereafter. Comparison of left vs. right atrial pacing in rats was also feasible with the current technique, resulting in similar AERP values recorded in the low right atrium. In conclusion, our findings indicate that in vivo rate adaptation of the rodent atria is different than expected based on previous ex vivo recordings. In addition, atrial electrical remodeling of rats shows unique remodeling-reverse remodeling characteristics that are described here for the first time. Further understanding of these properties should help to determine the clinical relevance as well as limitations of atrial arrhythmia models in rodents.