Exosomes from COVID-19 Patients Carry Tenascin-C and Fibrinogen-ß in Triggering Inflammatory Signals in Cells of Distant Organ.

SARS-CoV-2 infection can cause cytokine storm and may overshoot immunity in humans; however, it remains to be determined whether virus-induced soluble mediators from infected cells are carried by exosomes as vehicles to distant organs and cause tissue damage in COVID-19 patients. We took an unbiased proteomic approach for analyses of exosomes isolated from plasma of healthy volunteers and COVID-19 patients. Our results revealed that tenascin-C (TNC) and fibrinogen-ß (FGB) are highly abundant in exosomes from COVID-19 patients' plasma compared with that of healthy normal controls. Since TNC and FGB stimulate pro-inflammatory cytokines via the Nuclear factor-κB (NF-κB) pathway, we examined the status of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-C motif chemokine ligand 5 (CCL5) expression upon exposure of hepatocytes to exosomes from COVID-19 patients and observed significant increase compared with that from healthy subjects. Together, our results demonstrate that TNC and FGB are transported through plasma exosomes and potentially trigger pro-inflammatory cytokine signaling in cells of distant organ.

Momordicine-I, a Bitter Melon Bioactive Metabolite, Displays Anti-Tumor Activity in Head and Neck Cancer Involving c-Met and Downstream Signaling.

Head and neck cancer (HNC) is one of the most aggressive cancers, and treatments are quite challenging due to the difficulty in early diagnosis, lack of effective chemotherapeutic drugs, adverse side effects and therapy resistance. We identified momordicine-I (M-I), a bioactive secondary metabolite in bitter melon (Momordica charantia), by performing liquid chromatography-high resolution electrospray ionization mass spectrometry (LC-HRESIMS) analysis. M-I inhibited human HNC cell (JHU022, JHU029, Cal27) viability in a dose-dependent manner without an apparent toxic effect on normal oral keratinocytes. Mechanistic studies showed that M-I inhibited c-Met and its downstream signaling molecules c-Myc, survivin, and cyclin D1 through the inactivation of STAT3 in HNC cells. We further observed that M-I was non-toxic and stable in mouse (male C57Bl/6) blood, and a favorable pharmacokinetics profile was observed after IP administration. M-I treatment reduced HNC xenograft tumor growth in nude mice and inhibited c-Met and downstream signaling. Thus, M-I has potential therapeutic implications against HNC.

SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells.

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response.

Hemoglobin ß93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell.

BACKGROUND: Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin ß chain (ß93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. METHODS: To test this hypothesis, we used RBCs from mice in which the ß93 cysteine had been replaced with alanine (ß93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. RESULTS: In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (ß93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with ß93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in ß93C and ß93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between ß93C and ß93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in ß93C and ß93A mice. CONCLUSIONS: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the ß93 cysteine of Hb.

Bitter melon extract inhibits breast cancer growth in preclinical model by inducing autophagic cell death.

Breast cancer is a major public health problem worldwide in women and current therapeutic strategies are not adequately effective for this deadly disease. We have previously shown the anti-proliferative activity of bitter melon extract (BME) in breast cancer cells. In this study, we observed that BME treatment induces autophagosome-bound Long chain 3 (LC3)-B and accumulates protein p62/SQSTM1 (p62) in breast cancer cells. Additionally, we observed that BME treatment in breast cancer cells increases phospho-AMPK expression and inhibits the mTOR/Akt signaling pathway. Subsequently, we demonstrated that BME feeding effectively inhibited breast cancer growth in syngeneic and xenograft mouse models. Further, we observed the increased p62 accumulation, induction of autophagy and apoptotic cell death in tumors from BME-fed animals. Taken together, our results demonstrate that BME treatment inhibits breast tumor growth, and this anti-tumor activity in breast cancer is, in part, mediated by induction of autophagy and modulation of the AMPK/mTOR pathway. The antitumor activity of BME by oral feeding in breast cancer models suggested the high potential for a clinical application.

Cigarette smoke-induced urothelial cell damage: potential role of platelet-activating factor.

Cigarette smoking is an environmental risk factor associated with a variety of pathologies including cardiovascular disease, inflammation, and cancer development. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory bladder disease with multiple etiological contributors and risk factors associated with its development, including cigarette smoking. Previously, we determined that cigarette smoking was associated with bladder wall accumulation of platelet activating factor (PAF), a potent inflammatory mediator that facilitates transendothelial cell migration of inflammatory cells from the circulation. PAF has been shown to reduce expression of tight junctional proteins which could ultimately lead to increased urothelial cell permeability. In this study, we observed that cigarette smoke extract (CSE) treatment of human urothelial cells increases PAF production and PAF receptor expression and reduces wound healing ability. After exposure to cigarette smoke for 6 months, wild-type C57BL/6 mice displayed urothelial thinning and destruction which was not detected in iPLA2ß-/- (enzyme responsible for PAF production) animals. We also detected increased urinary PAF concentration in IC/BPS patients when compared to controls, with an even greater increase in urinary PAF concentration in smokers with IC/BPS These data indicate that cigarette smoking is associated with urothelial cell damage that may be a result of increased PAF-PAF receptor interaction. Inhibition of iPLA2ß activity or blocking of the PAF-PAF receptor interaction could serve as a potential therapeutic target for managing cigarette smoke-induced bladder damage.

Hydrogen sulfide mediates the vasoactivity of garlic.

The consumption of garlic is inversely correlated with the progression of cardiovascular disease, although the responsible mechanisms remain unclear. Here we show that human RBCs convert garlic-derived organic polysulfides into hydrogen sulfide (H(2)S), an endogenous cardioprotective vascular cell signaling molecule. This H(2)S production, measured in real time by a novel polarographic H(2)S sensor, is supported by glucose-maintained cytosolic glutathione levels and is to a large extent reliant on reduced thiols in or on the RBC membrane. H(2)S production from organic polysulfides is facilitated by allyl substituents and by increasing numbers of tethering sulfur atoms. Allyl-substituted polysulfides undergo nucleophilic substitution at the alpha carbon of the allyl substituent, thereby forming a hydropolysulfide (RS(n)H), a key intermediate during the formation of H(2)S. Organic polysulfides (R-S(n)-R'; n > 2) also undergo nucleophilic substitution at a sulfur atom, yielding RS(n)H and H(2)S. Intact aorta rings, under physiologically relevant oxygen levels, also metabolize garlic-derived organic polysulfides to liberate H(2)S. The vasoactivity of garlic compounds is synchronous with H(2)S production, and their potency to mediate relaxation increases with H(2)S yield, strongly supporting our hypothesis that H(2)S mediates the vasoactivity of garlic. Our results also suggest that the capacity to produce H(2)S can be used to standardize garlic dietary supplements.

Effects of sodium nitrite on ischemia-reperfusion injury in the rat kidney.

Reactive oxygen and nitrogen species play a key role in the pathophysiology of renal ischemia-reperfusion (I/R) injury. Recent studies have shown that nitrite (NO(2)(-)) serves as an endogenous source of nitric oxide (NO), particularly in the presence of hypoxia and acidosis. Nanomolar concentrations of NO(2)(-) reduce injury following I/R in the liver and heart in vivo. The purpose of this study was to evaluate the role of NO(2)(-) in renal I/R injury. Male Sprague-Dawley rats underwent a unilateral nephrectomy followed by 45 min of ischemia of the contralateral kidney or sham surgery under isoflurane anesthesia. Animals received normal saline, sodium NO(2)(-), or sodium nitrate (NO(3)(-); 1.2 nmol/g body wt ip) at 22.5 min after induction of ischemia or 15 min before ischemia. A separate set of animals received saline, NO(2)(-), or NO(3)(-) (0.12, 1.2, or 12 nmol/g body wt iv) 45 min before ischemia. Serum creatinine and blood urea nitrogen were increased following I/R injury but were not significantly different among treatment groups at 24 and 48 h after acute renal injury. Interestingly, NO(3)(-) administration appeared to worsen renal injury. Histological scoring for loss of brush border, tubular necrosis, and red blood cell extravasation showed no significant differences among the treatment groups. The results indicate that, contrary to the protective effects of NO(2)(-) in I/R injury of the liver and heart, NO(2)(-) does not provide protection in renal I/R injury and suggest a unique metabolism of NO(2)(-) in the kidney.

Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation.

Local vasodilation in response to hypoxia is a fundamental physiologic response ensuring oxygen delivery to tissues under metabolic stress. Recent studies identify a role for the red blood cell (RBC), with hemoglobin the hypoxic sensor. Herein, we investigate the mechanisms regulating this process and explore the relative roles of adenosine triphosphate, S-nitrosohemoglobin, and nitrite as effectors. We provide evidence that hypoxic RBCs mediate vasodilation by reducing nitrite to nitric oxide (NO) and ATP release. NO dependence for nitrite-mediated vasodilation was evidenced by NO gas formation, stimulation of cGMP production, and inhibition of mitochondrial respiration in a process sensitive to the NO scavenger C-PTIO. The nitrite reductase activity of hemoglobin is modulated by heme deoxygenation and heme redox potential, with maximal activity observed at 50% hemoglobin oxygenation (P(50)). Concomitantly, vasodilation is initiated at the P(50), suggesting that oxygen sensing by hemoglobin is mechanistically linked to nitrite reduction and stimulation of vasodilation. Mutation of the conserved beta93cys residue decreases the heme redox potential (ie, decreases E(1/2)), an effect that increases nitrite reductase activity and vasodilation at any given hemoglobin saturation. These data support a function for RBC hemoglobin as an allosterically and redox-regulated nitrite reductase whose "enzyme activity" couples hypoxia to increased NO-dependent blood flow.