NETosis in Acute Thrombotic Disorders.

The release of extracellular traps by neutrophils (NETs) represents a novel active mechanism of cell death that has been recently implicated in the pathogenesis of thrombotic disorders. The aim of this study was to investigate the generation of NETs in different groups of patients with acute thrombotic events (ATEs) and to establish whether NETs markers can predict the risk of new cardiovascular events. We performed a case-control study of patients with ATE, including acute coronary syndrome (n = 60), cerebrovascular accident (n = 50), and venous thromboembolism (n = 55). Control subjects (n = 70) were identified among patients admitted for acute chest pain and in which a diagnosis of ATE was excluded. Serum levels of NET markers and neutrophil activation, such as myeloperoxidase (MPO)-DNA complexes, neutrophil gelatinase-associated lipocalin, polymorphonuclear neutrophil elastase, lactoferrin, and MPO, were measured in each patient. We found that circulating levels of MPO-DNA complexes were significantly increased in patients with ATE (p < 0.001) compared with controls and that this association remained significant even after fully adjustment for traditional risk factors (p = 0.001). A receiver operating characteristics analysis of circulating MPO-DNA complexes in discriminating between controls and patients with ATE showed a significant area under the curve of 0.76 (95% confidence interval: 0.69-0.82). After a median follow-up of 40.7 (± 13.8) months, 24 out of the 165 patients with ATE presented a new cardiovascular event and 18 patients died. None of the markers under investigation influenced survival or the incidence of new cardiovascular events. In conclusion, we found that increase of markers of NETosis can be observed in acute thrombotic conditions, occurring both on the arterial and venous site. Nevertheless, the level of neutrophil markers measured during the ATE is not predictive of future risk of mortality and cardiovascular events.

A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels.

Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca2+) uptake with a Ca2+-selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca2+ concentration ([Ca2+]cyt) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues.

Subclinical atherosclerosis evolution during 5 years of anti-TNF-alpha treatment in psoriatic arthritis patients.

OBJECTIVES: Our aim was to evaluate subclinical atherosclerosis progression during 5 years of anti-tumour necrosis factor (TNF)-α treatment in psoriatic arthritis (PsA) patients. METHODS: Thirty-two consecutive PsA patients starting TNF-α inhibitors were enrolled and evaluated at baseline (T0), 2 years (FU1) and 5 years (FU2) of treatment. Arterial structural properties were evaluated by B-mode ultrasound of mean carotid intima-media thickness (mean-IMT) and maximum IMT (M-MAX) in each segment (common, bulb, internal), bilaterally. Endothelial function was assessed by post-occlusion flow-mediated dilation (FMD) of the brachial artery using high-sensitivity ultrasonography. Treatment response was studied through DAS28 (disease activity score) and inflammatory biomarkers (C-reactive protein, TNF-α, osteoprotegerin). Metrologic and metabolic data were collected. RESULTS: At T1, a significant decrease of DAS28 (4.2±0.7 vs. 2.3±0.8, p<0.001) and CRP (11.25±9.16 vs. 2.91±1.72, p<0.01) was observed. Efficacy was preserved at FU2 (DAS28 2.4±0.9, CRP 2.73±2.51; p=ns vs. FU1). Systolic blood pressure and BMI remained stable throughout the follow-up, while diastolic blood pressure decreased significantly from FU1 to FU2 (80±10 vs. 74±7 mmHg, p=0.001). From T0 to FU1 there was an increase of IMT-mean and M-MAX (0.7±0.1 vs. 0.9±0.4 and 0.9±0.2 vs. 1.1±0.4, p<0.01). At FU2, IMT-mean and M-max did not change significantly (0.9±0.3 and 1.1±0.3, p=ns vs. FU1). No significant variation in FMD values was observed during the study period. CONCLUSIONS: A slight progression of subclinical atherosclerosis in PsA was observed in the first 2 years of anti-TNF-α treatment. This process seemed to decelerate in follow-up extension to 5 years.

Measurement of Ca2+ Uptake Through Connexin Hemichannels.

Increasing evidence points to deregulated flux of ionized calcium (Ca2+) mediated by hyperactive mutant connexin (Cx) hemichannels (HCs) as a common gain-of-function etiopathogenetic mechanism for several diseases, ranging from skin disorders to nervous system defects. Furthermore, the opening of nonmutated Cx HCs is associated with an impressive list of widespread diseases including, but not limited to, ischemia/stroke, Alzheimer's disease, and epilepsy. HC inhibitors are attracting a growing attention due to their therapeutic potential for numerous pathologies. This chapter describes a quantitative method to measure Ca2+ uptake though HCs expressed in cultured cells. The assay we developed can be used to probe HC activity as wells as to test HC inhibitors. Furthermore, with minor changes it can be easily adapted to high-throughput high-content platforms and/or primary cells and microtissues.

Antibody gene transfer treatment drastically improves epidermal pathology in a keratitis ichthyosis deafness syndrome model using male mice.

BACKGROUND: Keratitis ichthyosis deafness (KID) syndrome is a rare disorder caused by hemichannel (HC) activating gain-of-function mutations in the GJB2 gene encoding connexin (Cx) 26, for which there is no cure, or current treatments based upon the mechanism of disease causation. METHODS: We applied Adeno Associated Virus (AAV) mediated mAb gene transfer (AAVmAb) to treat the epidermal features of KID syndrome with a well-characterized HC blocking antibody using male mice of a murine model that replicates the skin pathology of the human disease. FINDINGS: We demonstrate that in vivo AAVmAb treatment significantly reduced the size and thickness of KID lesions, in addition to blocking activity of mutant HCs in the epidermis in vivo. We also show that AAVmAb treatment eliminated abnormal keratinocyte proliferation and enlarged cell size, decreased apoptosis, and restored the normal distribution of keratin expression. INTERPRETATION: Our findings reinforce the critical role played by increased HC activity in the skin pathology associated with KID syndrome. They also underscore the clinical potential of anti-HC mAbs coupled with genetic based delivery systems for treating the underlying mechanistic basis of this disorder. Inhibition of HC activity is an ideal therapeutic target in KID syndrome, and the genetic delivery of mAbs targeted against mutant HCs could form the basis of new therapeutic interventions to treat this incurable disease. FUNDING: Fondazione Telethon grant GGP19148 and University of Padova grant Prot. BIRD187130 to FM; Foundation for Ichthyosis and Related Skin Types (FIRST) and National Institutes of Health grant EY 026911 to TWW.

Commercially derived versatile optical architecture for two-photon STED, wavelength mixing and label-free microscopy.

Multimodal microscopy combines multiple non-linear techniques that take advantage of different optical processes to generate contrast and increase the amount of information that can be obtained from biological samples. However, the most advanced optical architectures are typically custom-made and often require on-site adjustment of optical components performed by trained personnel for optimal performance. Here, we describe a hybrid system we built based on a commercial upright microscope. We show that our multimodal imaging platform can be used to seamlessly perform two-photon STED, wavelength mixing and label-free microscopy in both ex vivo and in vivo turbid samples. The system is stable and endowed with remote alignment hardware that ensures long-term operability also for non-expert users, using the alignment protocol described in this article and in the related material. This optical architecture is an important step forward towards a wider practical applicability of non-linear optics to bioimaging.

Calcium Signaling in the Photodamaged Skin: In Vivo Experiments and Mathematical Modeling.

The epidermis forms an essential barrier against a variety of insults. The overall goal of this study was to shed light not only on the effects of accidental epidermal injury, but also on the mechanisms that support laser skin resurfacing with intra-epidermal focal laser-induced photodamage, a widespread medical practice used to treat a range of skin conditions. To this end, we selectively photodamaged a single keratinocyte with intense, focused and pulsed laser radiation, triggering Ca2+ waves in the epidermis of live anesthetized mice with ubiquitous expression of a genetically encoded Ca2+ indicator. Waves expanded radially and rapidly, reaching up to eight orders of bystander cells that remained activated for tens of minutes, without displaying oscillations of the cytosolic free Ca2+ concentration ([Formula: see text]). By combining in vivo pharmacological dissection with mathematical modeling, we demonstrate that Ca2+ wave propagation depended primarily on the release of ATP, a prime damage-associated molecular patterns (DAMPs), from the hit cell. Increments of the [Formula: see text] in bystander cells were chiefly due to Ca2+ release from the endoplasmic reticulum (ER), downstream of ATP binding to P2Y purinoceptors. ATP-dependent ATP release though connexin hemichannels (HCs) affected wave propagation at larger distances, where the extracellular ATP concentration was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases, whereas pannexin channels had no role. Bifurcation analysis suggests basal keratinocytes have too few P2Y receptors (P2YRs) and/or phospholipase C (PLC) to transduce elevated extracellular ATP levels into inositol trisphosphate (IP3) production rates sufficiently large to sustain [Formula: see text] oscillations.

Lipid Profile and Vascular Remodelling in Young Dyslipidemic Subjects Treated with Nutraceuticals Derived from Red Yeast Rice.

BACKGROUND AND AIMS: A relevant role is emerging for functional foods in cardiovascular prevention. The aim of this study was to assess the effect of a nutraceutical multitargeted approach on lipid profile and inflammatory markers along with vascular remodelling in a cohort of dyslipidemic subjects without history of cardiovascular (CV) disease. METHODS AND RESULTS: We enrolled 25 subjects (mean age 48.2 years) with low to moderate CV risk profile and total cholesterol (TC) levels between 150 and 250 mg/dl. The patients were assigned to receive for one year a tablet/die of a nutraceutical combination containing red yeast rice (RYR) extract (Monacolin 3 mg/tablet) and coenzyme Q10 (30 mg/tablet). Treatment with the nutraceutical compounds led to a significant reduction of TC (from 227 to 201 mg/dl, p < 0.001), LDL-c (from 150 to 130 mg/dl, p = 0.001), triglycerides (from 121 to 109 mg/dl, p = 0.013), non-HDL-cholesterol (from 168 to 141 mg/dl, p < 0.001), hs-CRP (from 1.74 to 1.20 mg/l, p = 0.015), and osteoprotegerin (from 1488 to 1328 pg/ml, p = 0.045). Levels of HDL-c, Lp(a), glucose, liver enzyme, CPK, or creatinine did not change over time. An ultrasound study was performed to assess changes in mean carotid intima-media thickness (IMT) and maximum IMT (M-MAX) as well as modification in local carotid stiffness by means of determining the carotid compliance coefficient (CC) and distensibility coefficient (DC). At the end of the treatment, we observed small but significant reductions in both mean-IMT (from 0.62 to 0.57 mm, p = 0.022) and M-MAX (from 0.79 to 0.73 mm, p = 0.002), and an improvement in carotid elasticity (DC from 22.4 to 24.3 × 10-3/kPa, p = 0.006 and CC from 0.77 to 0.85 mm2/kPa, p = 0.019). CONCLUSIONS: A long-term treatment with a combination of RYR and coenzyme Q10 showed lipid-lowering activity along with a reduction of inflammatory mediators and an improvement of vascular properties in young subjects with a low-to-moderate CV risk profile.

Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing.

In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT.

Serum uric acid levels and the risk of recurrent venous thromboembolism.

Essentials Increase in serum uric acid (SUA) levels has been widely associated with higher risk of cardiovascular disease. We investigated the link between SUA levels and the risk of venous thromboembolism (VTE) recurrence. Patients with SUA levels ≥ 4.38 mg/dL showed a three-fold increase in the risk of VTE recurrence. Elevated SUA levels are associated with increased risk of recurrent VTE independently from traditional risk factors. ABSTRACT: Background The link between serum uric acid (SUA) and the risk of cardiovascular disease is well established. However, the impact of SUA levels on the risk of venous thromboembolism (VTE) recurrence is unknown. Objectives To investigate the association between SUA and the risk of VTE recurrence. Patients and Methods We performed a monocenter, prospective study on 280 patients with a previous episode of VTE that completed the oral anticoagulant period. SUA levels at enrollment were correlated with the risk of VTE recurrence (mean follow-up 71.1 ± 29.2 months). Results Patients were stratified according to SUA tertiles distribution at baseline (tertiles cut-off: I ≤ 4.37 mg/dL, II 4.38--5.54 mg/dL, III ≥ 5.55 mg/dL). Fifty episodes of VTE recurrence occurred during the follow-up and Kaplan-Meier survival analysis showed that subjects in the lower tertile of SUA distribution had significantly lower risk of future VTE recurrence (P = .003). No differences were seen among patients belonging to the second and the third tertile of SUA distribution. A multivariate Cox regression analysis showed that higher tertiles of SUA distribution had about three-fold increase in the risk of VTE recurrence as compared to subjects with SUA ≤ 4.37, independently from potential confounders (hazard ratio [HR] 3.04, 95% confidence interval [CI] 1.15--8.05 P = .025). Moreover, we observed that the adjusted hazard of VTE recurrence increased by 30% for each additional unit of SUA (mg/dL; HR 1.30, 95% CI 1.01--1.22, P = .040). Conclusion Elevated SUA levels are associated with increased risk of future VTE recurrence independently from traditional risk factors.

Role of Vascular Adaptation in Determining Systolic Blood Pressure in Young Adults.

Background Two individuals can have a similar pulse pressure (PP) but different levels of systolic blood pressure (SBP), although the underlying mechanisms have not been described. We hypothesized that, for a given level of PP, differences in SBP relate to peripheral vascular resistance (PVR); and we tested this hypothesis in a large cohort of healthy young adults. Methods and Results Demographic, biochemical, and hemodynamic data from 3103 subjects were available for the current analyses. In both men and women, for a given level of PP, higher SBP was associated with significantly higher body weight, body mass index, heart rate, and PVR (P<0.05 versus those with lower BP for all comparisons). Moreover, stratifying individuals by quartiles of PP and PVR revealed a stepwise increase in SBP from the lowest to highest quartile for each variable, with the highest SBP occurring in those in the highest quartile of both PP and PVR (P<0.001 for overall trend for both sexes). PVR was also increased with increasing tertile of minimum forearm vascular resistance, in both men (P=0.002) and women (P=0.03). Conclusions Increased PVR, mediated in part through altered resistance vessel structure, strongly associates with the elevation of SBP for a given level of PP in young adults. An impaired ability to adapt PVR appropriately to a given level of PP may be an important mechanism underlying elevated SBP in young adults.

l-Arginine prevents inflammatory and pro-calcific differentiation of interstitial aortic valve cells.

BACKGROUND AND AIMS: Reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of calcific aortic stenosis. Herein, we investigated the effects of l-Arginine, the main precursor of NO, on the osteogenic differentiation of aortic interstitial valve cells (VICs). METHODS: We isolated a clonal population of bovine VICs that expresses osteogenic markers and induces calcification of collagen matrix after stimulation with endotoxin (LPS 500 ng/mL). VICs were treated in vitro with different combinations of LPS ± l-Arginine (50 or 100 mM) and cell extracts were collected to perform proteomic (iTRAQ) and gene expression (RT-PCR) analysis. RESULTS: l-Arginine prevents the over-expression of alkaline phosphatase (ALP, p < 0.001) and reduces matrix calcification (p < 0.05) in VICs treated with LPS. l-Arginine also reduces the over-expression of inflammatory molecules induced by LPS (TNF-alpha, IL-6 and IL-1beta, p < 0.001). The proteomic analysis allowed to identify 49 proteins with an altered expression profile after stimulation with LPS and significantly modified by l-Arginine. These include proteins involved in the redox homeostasis of the cells (i.e. Xanthine Oxidase, Catalase, Aldehyde Oxidase), remodeling of the extracellular matrix (i.e. ADAMTSL4, Basigin, COL3A1) and cellular signaling (i.e. Fibrillin-1, Legumain, S100A13). The RT-PCR analysis confirmed the modifications of Fibrillin-1, ADAMTSL4, Basigin and Xanthine Oxidase, whose expression levels increase after stimulation with LPS and are reduced by l-Arginine (p < 0.05). CONCLUSIONS: l-Arginine prevents osteogenic differentiation of VICs and reduces matrix calcification. This effect is achieved through the modulation of proteins involved in the cellular redox system, remodeling of extracellular matrix and inflammatory activation of VICs.

Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge in the developing cochlea.

Prior work supports the hypothesis that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge (GER) in the developing cochlea; however, direct proof is lacking. To address this issue, we plated cochlear organotypic cultures (COCs) and whole cell-based biosensors with nM ATP sensitivity (ATP-WCBs) at the bottom and top of an ad hoc designed transparent microfluidic chamber, respectively. By performing dual multiphoton Ca2+ imaging, we monitored the propagation of intercellular Ca2+ waves in the GER of COCs and ATP-dependent Ca2+ responses in overlying ATP-WCBs. Ca2+ signals in both COCs and ATP-WCBs were inhibited by supplementing the extracellular medium with ATP diphosphohydrolase (apyrase). Spontaneous Ca2+ signals were strongly depressed in the presence of Gjb6-/- COCs, in which connexin 30 (Cx30) is absent and connexin 26 (Cx26) is strongly downregulated. In contrast, spontaneous Ca2+ signals were not affected by replacement of Panx1-/- with Panx1+/+ COCs in the microfluidic chamber. Similar results were obtained by estimating ATP release from COCs using a classical luciferin-luciferase bioluminescence assay. Therefore, connexin hemichannels and not pannexin 1 channels mediate the release of ATP that is responsible for Ca2+ wave propagation in the developing mouse cochlea. The technological advances presented here have the potential to shed light on a plethora of unrelated open issues that involve paracrine signaling in physiology and pathology and cannot be addressed with standard methods.

Use of RAAS Inhibitors and Risk of Clinical Deterioration in COVID-19: Results From an Italian Cohort of 133 Hypertensives.

BACKGROUND: The effect of chronic use of renin-angiotensin-aldosterone system (RAAS) inhibitors on the severity of COVID-19 infection is still unclear in patients with hypertension. We aimed to investigate the association between chronic use of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs) and COVID-19-related outcomes in hypertensive patients. METHODS: A single-center study was conducted on 133 consecutive hypertensive subjects presenting to the emergency department with acute respiratory symptoms and/or fever who were diagnosed with COVID-19 infection between 9 and 31 March 2020. RESULTS: All patients were grouped according to their chronic antihypertensive medications (ACEIs, N = 40; ARBs, N = 42; not on RAAS inhibitors, N = 51). There was no statistical difference between ACEIs and ARBs groups in terms of hospital admission rate, oxygen therapy, and need for noninvasive ventilation. Patients chronically treated with RAAS inhibitors showed a significantly lower rate of admission to semi-intensive/intensive care units, when compared with the non-RAAS population (odds ratio (OR) 0.25, confidence interval (CI) 95% 0.09-0.66, P = 0.006). Similarly, the risk of mortality was lower in the former group, although not reaching statistical significance (OR 0.56, CI 95% 0.17-1.83, P = 0.341). CONCLUSIONS: Our data suggest that chronic use of RAAS inhibitors does not negatively affect clinical course of COVID-19 in hypertensive patients. Further studies are needed to confirm this finding and determine whether RAAS inhibitors may have a protective effect on COVID-19-related morbidity and mortality.

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice.

BACKGROUND: Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as "leaky" hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels. METHODS: We employed the antibody to treat Cx30A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca2+ imaging and ATP release assay in vitro. FINDINGS: Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels. INTERPRETATION: Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. FUND: Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.

Blood Pressure Variability and Therapeutic Implications in Hypertension and Cardiovascular Diseases.

Blood pressure (BP) is characterized by continuous dynamic and spontaneous oscillations occurring over lifetime and defining the so-called blood pressure variability (BPV). BPV has been associated with target organ damage, cardiovascular (CV) risk and death, suggesting the use of BPV as a new target in hypertension management in addition to mean BP values lowering. The purpose of the review is to focus on the therapeutic implications of BPV and summarize the effects of different drug classes on various types of BPV. Despite most first-line antihypertensive medications contribute to reduce both short and long term BPV, calcium channel blockers (CCBs) as monotherapy or fixed-combination therapy appear to be the most effective on BPV control. Further randomized interventional trials are needed to investigate which drug combinations are most appropriate according to patient CV risk stratification, in order to improve their CV outcomes.

Photosensitizer Activation Drives Apoptosis by Interorganellar Ca2+ Transfer and Superoxide Production in Bystander Cancer Cells.

In cells, photosensitizer (PS) activation by visible light irradiation triggers reactive oxygen species (ROS) formation, followed by a cascade of cellular responses involving calcium (Ca2+) and other second messengers, resulting in cell demise. Cytotoxic effects spread to nearby cells not exposed to light by poorly characterized so-called "bystander effects". To elucidate the mechanisms involved in bystander cell death, we used both genetically encoded biosensors and fluorescent dyes. In particular, we monitored the kinetics of interorganellar Ca2+ transfer and the production of mitochondrial superoxide anion (O2-∙) and hydrogen peroxide (H2O2) in irradiated and bystander B16-F10 mouse melanoma cancer cells. We determined that focal PS photoactivation in a single cell triggers Ca2+ release from the endoplasmic reticulum (ER) also in the surrounding nonexposed cells, paralleled by mitochondrial Ca2+ uptake. Efficient Ca2+ efflux from the ER was required to promote mitochondrial O2-∙ production in these bystander cells. Our results support a key role for ER-mitochondria communication in the induction of ROS-mediated apoptosis in both direct and indirect photodynamical cancer cell killing.

A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function.

Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a "leaky" character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro. Altogether, these results indicate abEC1.1 is a promising tool for further translational studies.

Warfarin, but not rivaroxaban, promotes the calcification of the aortic valve in ApoE-/- mice.

INTRODUCTION: Vitamin K antagonists, such as warfarin, are known to promote arterial calcification through blockade of gamma-carboxylation of Matrix-Gla-Protein. It is currently unknown whether other oral anticoagulants such as direct inhibitors of Factor Xa can have protective effects on the progression of aortic valve calcification. AIMS: To compare the effect of warfarin and rivaroxaban on the progression of aortic valve calcification in atherosclerotic mice. RESULTS: 42 ApoE-/- mice fed with Western-type Diet (WTD) were randomized to treatment with warfarin (n = 14), rivaroxaban (n = 14) or control (n = 14) for 8 weeks. Histological analyses were performed to quantify the calcification of aortic valve leaflets and the development of atherosclerosis. The analyses showed a significant increase in valve calcification in mice treated with warfarin as compared to WTD alone (P = .025) or rivaroxaban (P = .005), whereas no significant differences were found between rivaroxaban and WTD (P = .35). Quantification of atherosclerosis and intimal calcification was performed on the innominate artery of the mice and no differences were found between the 3 treatments as far as atherogenesis and calcium deposition is concerned. In vitro experiments performed using bovine interstitial valve cells (VIC) showed that treatment with rivaroxaban did not prevent the osteogenic conversion of the cells but reduce the over-expression of COX-2 induced by inflammatory mediators. CONCLUSION: We showed that warfarin, but not rivaroxaban, could induce calcific valve degeneration in a mouse model of atherosclerosis. Both the treatments did not significantly affect the progression of atherosclerosis. Overall, these data suggest a safer profile of rivaroxaban on the risk of cardiovascular disease progression.

Cardiovascular Phenotype of Elevated Blood Pressure Differs Markedly Between Young Males and Females: The Enigma Study

Blood pressure (BP) in young adults predicts BP in later life. We aimed to identify metabolic, hemodynamic, and autonomic characteristics associated with raised BP in young adults and whether these differ between males and females. Three thousand one hundred forty-five healthy subjects, aged 18 to 40 years, were grouped according to sex and BP category following the recent reclassification of BP as part of American Heart Association/American College of Cardiology 2017 guidelines. All individuals undertook a lifestyle and medical history questionnaire and detailed metabolic, hemodynamic, and autonomic assessments. Stage 1 hypertension and normal BP were the most common BP phenotypes in males (29%) and females (68%), respectively. In both sexes, cardiac output was positively associated with increasing BP category (P<0.001 for both). Similar positive trends were observed for heart rate and stroke volume in males (P<0.001 for both) and heart rate in females (P<0.001). Unlike in males, peripheral vascular resistance, aortic pulse wave velocity, and augmentation index were significantly increased in hypertensive females (P<0.001 for all) compared with the other BP categories. Most heart rate variability indices decreased across the BP categories, particularly in males. In young adults, metabolic and hemodynamic abnormalities associated with hypertension are already present at the elevated BP stage and the overall phenotype differed markedly between sexes. Whereas a cardiac phenotype was associated with elevated BP and hypertension in males, a vascular phenotype, characterized by elevated peripheral vascular resistance, aortic pulse wave velocity, and augmentation index, was dominant in females.