Assessment of ATP Metabolism to Adenosine by Ecto-Nucleotidases Carried by Tumor-Derived Small Extracellular Vesicles.

Background: Immunosuppression is a hallmark of cancer progression. Tumor-derived small extracellular vesicles (sEV), also known as TEX, produce adenosine (ADO) and can mediate tumor-induced immunosuppression. Methods: Here, the ATP pathway of ADO production (ATP◊ADP◊AMP◊ADO) by ecto-nucleotidases carried in sEV was evaluated by a novel method using N6-etheno-ATP (eATP) and N6-etheno-AMP (eAMP) as substrates. The "downstream" N6-etheno-purines (ePurines) were measured by high performance liquid chromatography with fluorescence detection (HPLC-FL). Results: Human melanoma cell-derived TEX (MTEX) metabolized eATP to N6-etheno-ADP (eADP), eAMP and N6-etheno-Adenosine (eADO) more robustly than control keratinocyte cell-derived sEV (CEX); due to accelerated conversion of eATP to eADP and eADP to eAMP MTEX and CEX similarly metabolized eAMP to eADO. Blocking of the ATP pathway with the selective CD39 inhibitor ARL67156 or pan ecto-nucleotidase inhibitor POM-1 normalized the ATP pathway but neither inhibitor completely abolished it. In contrast, inhibition of CD73 by PSB12379 or AMPCP abolished eADO formation in both MTEX and CEX, suggesting that targeting CD73 is the preferred approach to eliminating ADO produced by sEV. Conclusions: The noninvasive, sensitive, and specific assay assessing ePurine metabolism ± ecto-nucleotidase inhibitors in TEX enables the personalized identification of the ecto-nucleotidase primarily involved in ADO production in patients with cancer. The assay could guide precision medicine by determining which purine is the preferred target for inhibitory therapeutic interventions.

Purine nucleoside phosphorylase inhibition is an effective approach for the treatment of chemical hemorrhagic cystitis.

Hemorrhagic cystitis may be induced by infection, radiation therapy, or medications or may be idiopathic. Along with hemorrhagic features, symptoms include urinary urgency and frequency, dysuria (painful urination), and visceral pain. Cystitis-induced visceral pain is one of the most challenging types of pain to treat, and an effective treatment would address a major unmet medical need. We assessed the efficacy of a purine nucleoside phosphorylase inhibitor, 8-aminoguanine (8-AG), for the treatment of hemorrhagic/ulcerative cystitis. Lower urinary tract (LUT) function and structure were assessed in adult Sprague-Dawley rats, treated chronically with cyclophosphamide (CYP; sacrificed day 8) and randomized to daily oral treatment with 8-AG (begun 14 days prior to CYP induction) or its vehicle. CYP-treated rats exhibited multiple abnormalities, including increased urinary frequency and neural mechanosensitivity, reduced bladder levels of inosine, urothelial inflammation/damage, and activation of spinal cord microglia, which is associated with pain hypersensitivity. 8-AG treatment of CYP-treated rats normalized all observed histological, structural, biochemical, and physiological abnormalities. In cystitis 8-AG improved function and reduced both pain and inflammation likely by increasing inosine, a tissue-protective purine metabolite. These findings demonstrate that 8-AG has translational potential for reducing pain and preventing bladder damage in cystitis-associated LUT dysfunctions.

8-Aminopurines in the Cardiovascular and Renal Systems and Beyond.

Screening of compounds comprising 8-substituted guanine revealed that 8-aminoguanosine and 8-aminoguanine cause diuresis/natriuresis/glucosuria, yet decrease potassium excretion. Subsequent investigations demonstrated that 8-aminoguanosine's effects are mediated by its metabolite 8-aminoguanine. The mechanism by which 8-aminoguanine causes diuresis/natriuresis/glucosuria involves inhibition of PNPase (purine nucleoside phosphorylase), which increases renal interstitial inosine levels. Additional evidence suggests that inosine, via indirect or direct adenosine A2B receptor activation, increases renal medullary blood flow which enhances renal excretory function. Likely, 8-aminoguanine has pleiotropic actions that also alter renal excretory function. Indeed, the antikaliuretic effects of 8-aminoguanine are independent of PNPase inhibition. 8-Aminoguanine is an endogenous molecule; nitrosative stress leads to production of biomolecules containing 8-nitroguanine moieties. Degradation of these biomolecules releases 8-nitroguanosine and 8-nitro-2'-deoxyguanosine which are converted to 8-aminoguanine. Also, guanosine and guanine per se may contribute to 8-aminoguanine formation. 8-Aminoinosine, 8-aminohypoxanthine, and 8-aminoxanthine likewise induce diuresis/natriuresis/glucosuria, yet do not reduce potassium excretion. Thus, there are several pharmacologically active 8-aminopurines with nuanced effects on renal excretory function. Chronic treatment with 8-aminoguanine attenuates hypertension in deoxycorticosterone/salt rats, prevents strokes, and increases lifespan in Dahl salt-sensitive rats on a high salt diet and attenuates the metabolic syndrome in rats; 8-aminoguanosine retards progression of pulmonary hypertension in rats and anemia and organ damage in sickle cell mice. 8-Aminoguanine reverses age-associated lower urinary tract dysfunction and retinal degeneration. 8-Aminopurines represent a new class of agents (and potentially endogenous factors) that have beneficial effects on the cardiovascular system and kidneys and may turn back the clock in age-associated diseases.

Hypoxanthine Induces Signs of Bladder Aging with Voiding dysfunction and Lower Urinary Tract Remodeling.

BACKGROUND: Lower urinary tract syndrome (LUTS) is a group of urinary tract symptoms and signs which can include urinary incontinence. Advancing age is a major risk factors for LUTS; however the underlying biochemical mechanisms of age-related LUTS remain unknown. HX (hypoxanthine) is a purine metabolite associated with generation of tissue damaging reactive oxygen species (ROS). This study tested the hypothesis that exposure of the adult bladder to HX-ROS over time damages key LUT elements, mimicking qualitatively some of the changes observed with aging. METHODS: Adult 3-month-old female Fischer 344 (F344) rats were treated with vehicle or HX (10 mg/kg/day; 3 weeks) administered in drinking water. Targeted purine metabolomics and molecular approaches were used to assess purine metabolites and biomarkers for oxidative stress and cellular damage. Biomechanical approaches assessed LUT structure and measurements of LUT function (using custom-metabolic cages and cystometry) were also employed. RESULTS: HX exposure increased biomarkers indicative of oxidative stress, pathophysiological ROS production and depletion of cellular energy with declines in NAD + levels. Moreover, HX treatment caused bladder remodeling and decreased the intercontraction interval and leak point pressure (surrogate measure to assess stress urinary incontinence). CONCLUSIONS: These studies provide evidence that in adult rats chronic exposure to HX causes changes in voiding behavior and in bladder structure resembling alterations observed with aging. These results suggest that increased levels of uro-damaging HX were associated with ROS/oxidative stress-associated cellular damage which may be central to age-associated development of LUTS, opening up potential opportunities for geroscience-guided interventions.

8-Aminoguanine and Its Actions on Renal Excretory Function.

BACKGROUND: The endogenous purine 8-aminoguanine induces diuresis/natriuresis/glucosuria by inhibiting PNPase (purine nucleoside phosphorylase); however, mechanistic details are unknown. METHODS: Here, we further explored in rats 8-aminoguanine's effects on renal excretory function by combining studies using intravenous 8-aminoguanine, intrarenal artery infusions of PNPase substrates (inosine and guanosine), renal microdialysis, mass spectrometry, selective adenosine receptor ligands, adenosine receptor knockout rats, laser doppler blood flow analysis, cultured renal microvascular smooth muscle cells, HEK293 cells expressing A2B receptors and homogeneous time resolved fluorescence assay for adenylyl cyclase activity. RESULTS: Intravenous 8-aminoguanine caused diuresis/natriuresis/glucosuria and increased renal microdialysate levels of inosine and guanosine. Intrarenal inosine, but not guanosine, exerted diuretic/natriuretic/glucosuric effects. In 8-aminoguanine-pretreated rats, intrarenal inosine did not induce additional diuresis/natriuresis/glucosuria. 8-Aminoguanine did not induce diuresis/natriuresis/glucosuria in A2B-receptor knockout rats, yet did so in A1- and A2A-receptor knockout rats. Inosine's effects on renal excretory function were abolished in A2B knockout rats. Intrarenal BAY 60-6583 (A2B agonist) induced diuresis/natriuresis/glucosuria and increased medullary blood flow. 8-Aminoguanine increased medullary blood flow, a response blocked by pharmacological inhibition of A2B, but not A2A, receptors. In HEK293 cells expressing A2B receptors, inosine activated adenylyl cyclase, and this was abolished by MRS 1754 (A2B antagonist). In renal microvascular smooth muscle cells, 8-aminoguanine and forodesine (PNPase inhibitor) increased inosine and 3',5'-cAMP; however, in cells from A2B knockout rats, 8-aminoguanine and forodesine did not augment 3',5'-cAMP yet increased inosine. CONCLUSIONS: 8-Aminoguanine induces diuresis/natriuresis/glucosuria by increasing renal interstitial levels of inosine which, via A2B receptor activation, increases renal excretory function, perhaps in part by increasing medullary blood flow.

Dimethyl Sulfoxide Induces Hemolysis and Pulmonary Hypertension.

Vascular and lung injury are well established complications associated with hemolytic disorders, and hemolysis associated pulmonary hypertension (PH) has emerged as the most serious complication of sickle cell disease. The causal relationship between intravascular hemolysis and the development of PH is still under investigation. Previously we have shown that repetitive administration of hemolyzed autologous blood causes PH in rats. Dimethyl sulfoxide (DMSO), a widely used solvent and anti-inflammatory agent, induces hemolysis in vivo. We hypothesized that repetitive administration of DMSO would induce PH in rats. We also examined hemolysis-induced release of adenosine deaminase (ADA) and arginase from red blood cells, which may amplify hemolysis-mediated vascular injury. Acute administration of DMSO (1.5ml/30 min into the right atrium) induced intravascular hemolysis and pulmonary vasoconstriction. DMSO-induced increase in right ventricular peak systolic pressure (RVPSP) was associated with increased release of ADA. Notably, the acute increase in RVPSP was attenuated by administration of an adenosine A2A receptor agonist or by pretreatment of animals with ADA inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). Repetitive administration of DMSO for 10 days produced anemia, hemoglobinuria, hemoglobinemia, splenomegaly, and development of PH. Histopathological analysis revealed pulmonary vascular remodeling. The presented data describe a new model of hemolysis induced PH, suggesting that hemolysis is mechanistically related to pulmonary hypertension, and pointing to a potential pathogenic role that adenosine deaminase and accelerated adenosine metabolism may play in hemolysis associated pulmonary hypertension.

Suppressed renoprotective purines in COVID-19 patients with acute kidney injury.

Acute kidney injury (AKI) is common in patients hospitalized for COVID-19, complicating their clinical course and contributing to worse outcomes. Animal studies show that adenosine, inosine and guanosine protect the kidney against some types of AKI. However, until now there was no evidence in patients supporting the possibility that abnormally low kidney levels of adenosine, inosine and guanosine contribute to AKI. Here, we addressed the question as to whether these renoprotective purines are altered in the urine of COVID-19 patients with AKI. Purines were measured by employing ultra-high-performance liquid chromatography-tandem mass spectrometry with stable-isotope-labeled internal standards for each purine of interest. Compared with COVID-19 patients without AKI (n = 23), COVID-19 patients with AKI (n = 20) had significantly lower urine levels of adenosine (P < 0.0001), inosine (P = 0.0008), and guanosine (P = 0.0008) (medians reduced by 85%, 48% and 61%, respectively) and lower levels (P = 0.0003; median reduced by 67%) of the 2nd messenger for A2A and A2B adenosine receptors, i.e., 3',5'-cAMP. Moreover, in COVID-19 patients with AKI, urine levels of 8-aminoguanine (endogenous inhibitor of inosine and guanosine metabolism) were nearly abolished (P < 0.0001). In contrast, the "upstream" precursors of renoprotective purines, namely 5'-AMP and 5'-GMP, were not significantly altered in COVID-19 patients with AKI, suggesting defective conversion of these precursors by CD73 (converts 5'-AMP to adenosine and 5'-GMP to guanosine). These findings imply that an imbalance in renoprotective purines may contribute to AKI in COVID-19 patients and that pharmacotherapy targeted to restore levels of renoprotective purines may attenuate the risk of AKI in susceptible patients with COVID-19.

Purine nucleoside phosphorylase as a target to treat age-associated lower urinary tract dysfunction.

The lower urinary tract (LUT), including the bladder, urethra and external striated muscle, becomes dysfunctional with age; consequently, many older individuals suffer from lower urinary tract disorders (LUTDs). By compromising urine storage and voiding, LUTDs degrade quality of life for millions of individuals worldwide. Treatments for LUTDs have been disappointing, frustrating both patients and their physicians; however, emerging evidence suggests that partial inhibition of the enzyme purine nucleoside phosphorylase (PNPase) with 8-aminoguanine (an endogenous PNPase inhibitor that moderately reduces PNPase activity) reverses age-associated defects in the LUT and restores the LUT to that of a younger state. Thus, 8-aminoguanine improves LUT biochemistry, structure and function by rebalancing the LUT purine metabolome, making 8-aminoguanine a novel potential treatment for LUTDs.

Cargo and Functional Profile of Saliva-Derived Exosomes Reveal Biomarkers Specific for Head and Neck Cancer.

Background: Exosomes contribute to immunosuppression in head and neck squamous cell carcinoma (HNSCC), a tumor entity which lacks specific tumor biomarkers. Plasma-derived exosomes from HNSCC patients correlate with clinical parameters and have potential as liquid biopsy. Here, we investigate the cargo and functional profile of saliva-derived exosomes from HNSCC patients and their potential as non-invasive biomarkers for disease detection and immunomodulation. Methods: Exosomes were isolated from saliva of HNSCC patients (n = 21) and healthy donors (HD, n = 12) by differential ultracentrifugation. Surface values of immune checkpoints and tumor associated antigens on saliva-derived exosomes were analyzed by bead-based flow cytometry using CD63 capture. Upon co-incubation with saliva-derived exosomes, activity and proliferation of T cells were assessed by flow cytometry (CD69 expression, CFSE assay). Adenosine levels were measured by mass spectrometry after incubation of saliva-derived exosomes with exogenous ATP. miRNA profiling of saliva-derived exosomes was performed using the nCounter® SPRINT system. Results: Saliva-derived, CD63-captured exosomes from HNSCC patients carried high amounts of CD44v3, PDL1 and CD39. Compared to plasma, saliva was rich in tumor-derived, CD44v3+ exosomes and poor in hematopoietic cell-derived, CD45+ exosomes. CD8+ T cell activity was attenuated by saliva-derived exosomes from HNSCC patients, while proliferation of CD4+ T cells was not affected. Further, saliva-derived exosomes produced high levels of immunosuppressive adenosine. 62 HD- and 31 HNSCC-exclusive miRNAs were identified. Samples were grouped in "Healthy" and "Cancer" based on their saliva-derived exosomal miRNA profile, which was further found to be involved in RAS/MAPK, NF-κB complex, Smad2/3, and IFN-α signaling. Conclusions: Saliva-derived exosomes from HNSCC patients were enriched in tumor-derived exosomes whose cargo and functional profile reflected an immunosuppressive TME. Surface values of CD44v3, PDL1 and CD39 on CD63-captured exosomes, adenosine production and the miRNA cargo of saliva-derived exosomes emerged as discriminators of disease and emphasized their potential as liquid biomarkers specific for HNSCC.

Biochemical pathways of 8-aminoguanine production in Sprague-Dawley and Dahl salt-sensitive rats.

BACKGROUND: 8-Aminoguanine exerts natriuretic and antihypertensive activity. Whether and how "free" 8-aminoguanine exists in vivo is unclear. Because 8-nitroguanosine is naturally occurring, we tested the hypothesis that 8-aminoguanine can arise from: pathway 1, 8-nitroguanosine â†’ 8-aminoguanosine â†’ 8-aminoguanine; and pathway 2, 8-nitroguanosine â†’ 8-nitroguanine â†’ 8-aminoguanine. METHODS: 8-Aminoguanine biosynthesis was explored in rats using renal microdialysis, mass spectrometry and enzyme kinetics. RESULTS: In Sprague-Dawley rats, 8-nitroguanosine infusions increased kidney levels of 8-nitroguanine, 8-aminoguanosine and 8-aminoguanine; 8-nitroguanine infusions increased 8-aminoguanine. Purine nucleoside phosphorylase (PNPase) converted 8-nitroguanosine to 8-nitroguanine and 8-aminoguanosine to 8-aminoguanine. Forodesine (PNPase inhibitor) reduced metabolism of 8-nitroguanosine by pathway 2 and shunted metabolism of 8-nitroguanosine to 8-aminoguanosine. In Dahl salt-sensitive rats, 8-nitroguanosine infusions increased kidney levels of 8-nitroguanine, 8-aminoguanosine and 8-aminoguanine. These results indicate that both pathways 1 and 2 participate in the biosynthesis of 8-aminoguanine in Sprague-Dawley and Dahl rats. Endogenous 8-aminoguanine in kidneys and urine were elevated many-fold in Dahl, compared to Sprague-Dawley, rats. The increased levels of 8-aminoguanine in Dahl rats were not due to alterations in pathways 1 and 2 but were associated with increased urine levels of endogenous 8-nitroguanosine suggesting that the "upstream" production of 8-nitroguanosine was increased in Dahl rats. Dahl rats are known to have high levels of peroxynitrite, and peroxynitrite is known to nitrate guanosine in biomolecules. Here we confirm that a peroxynitrite donor increases kidney levels of 8-aminoguanine. CONCLUSION: 8-Aminoguanine occurs naturally via two distinct pathways and kidney levels of 8-aminoguanine are increased in Dahl rats, likely due to increased production of 8-nitroguanosine, a by-product of peroxynitrite chemistry.

8-Aminoinosine and 8-Aminohypoxanthine Inhibit Purine Nucleoside Phosphorylase and Exert Diuretic and Natriuretic Activity.

8-Aminoguanine and 8-aminoguanosine (via metabolism to 8-aminoguanine) are endogenous 8-aminopurines that induce diuresis, natriuresis, and glucosuria by inhibiting purine nucleoside phosphorylase (PNPase); moreover, both 8-aminopurines cause antikaliuresis by other mechanisms. Because 8-aminoinosine and 8-aminohypoxanthine are structurally similar to 8-aminoguanosine and 8-aminoguanine, respectively, we sought to define their renal excretory effects. First, we compared the ability of 8-aminoguanine, 8-aminohypoxanthine, and 8-aminoinosine to inhibit recombinant PNPase. These compounds inhibited PNPase with a potency order of 8-aminoguanine > 8-aminohypoxanthine = 8-aminoinosine. Additional studies showed that 8-aminoinosine is a competitive substrate that is metabolized to a competitive PNPase inhibitor, namely 8-aminohypoxanthine. Administration of each 8-aminopurine (33.5 µmol/kg) reduced the guanine-to-guanosine and hypoxanthine-to-inosine ratios in urine, a finding confirming their ability to inhibit PNPase in vivo. All three 8-aminopurines induced diuresis, natriuresis, and glucosuria; however, the glucosuric effects of 8-aminohypoxanthine and 8-aminoinosine were less pronounced than those of 8-aminoguanine. Neither 8-aminohypoxanthine nor 8-aminoinosine altered potassium excretion, whereas 8-aminoguanine caused antikaliuresis. In vivo administration of 8-aminoinosine increased 8-aminohypoxanthine excretion, indicating that 8-aminohypoxanthine mediates, in part, the effects of 8-aminoinosine. Finally, 8-aminohypoxanthine was metabolized to 8-aminoxanthine by xanthine oxidase. Using ultraperformance liquid chromatography-tandem mass spectrometry, we identified 8-aminoinosine as an endogenous 8-aminopurine. In conclusion, 8-aminopurines have useful pharmacological profiles. To induce diuresis, natriuresis, glucosuria, and antikaliuresis, 8-aminoguanine (or its prodrug 8-aminoguanosine) would be preferred. If only diuresis and natriuresis, without marked glucosuria or antikaliuresis, is desired, 8-aminohypoxanthine or 8-aminoinosine might be useful. Finally, here we report the in vivo existence of another pharmacologically active 8-aminopurine, namely 8-aminoinosine. SIGNIFICANCE STATEMENT: Here, we report that a family of 8-aminopurines affects renal excretory function: effects that may be useful for treating multiple diseases including hypertension, heart failure, and chronic kidney disease. For diuresis and natriuresis accompanied by glucosuria and antikaliuresis, 8-aminoguanine (or its prodrug 8-aminoguanosine) would be useful; if only diuresis and natriuresis is called for, 8-aminohypoxanthine or 8-aminoinosine would be useful. Previously, we identified 8-aminoguanine and 8-aminoguanosine as endogenous 8-aminopurines; here, we extend the family of endogenous 8-aminopurines to include 8-aminoinosine.

Angiotensin II augments renal vascular smooth muscle soluble GC expression via an AT1 receptor-forkhead box subclass O transcription factor signalling axis.

BACKGROUND AND PURPOSE: Reduced renal blood flow triggers activation of the renin-angiotensin-aldosterone system (RAAS) leading to renovascular hypertension. Renal vascular smooth muscle expression of the NO receptor, soluble GC (sGC), modulates the vasodilator response needed to control renal vascular tone and blood flow. Here, we tested if angiotensin II (Ang II) affects sGC expression via an AT1 receptor-forkhead box subclass O (FoxO) transcription factor dependent mechanism. EXPERIMENTAL APPROACH: Using a murine two-kidney-one-clip (2K1C) renovascular hypertension model, we measured renal artery vasodilatory function and sGC expression. Additionally, we conducted cell culture studies using rat renal pre-glomerular smooth muscle cells (RPGSMCs) to test the in vitro mechanistic effects of Ang II treatment on sGC expression and downstream function. KEY RESULTS: Contralateral, unclipped renal arteries in 2K1C mice showed increased NO-dependent vasorelaxation compared to sham control mice. Immunofluorescence studies revealed increased sGC protein expression in 2K1C contralateral renal arteries over sham controls. RPGSMCs treated with Ang II caused a significant up-regulation of sGC mRNA and protein expression as well as downstream sGC-dependent signalling. Ang II signalling effects on sGC expression occurred through an AT1 receptor and FoxO transcription factor-dependent mechanism at both the mRNA and protein expression levels. CONCLUSION AND IMPLICATIONS: Renal artery smooth muscle, in vivo and in vitro, up-regulates expression of sGC following RAAS activity. In both cases, up-regulation of sGC leads to increased downstream cGMP signalling, suggesting a previously unrecognized protective mechanism to improve renal blood flow in the uninjured contralateral renal artery. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Dysregulated Purine Metabolism Contributes to Age-Associated Lower Urinary Tract Dysfunctions.

Lower urinary tract (LUT) dysfunction is common in the older adult. Aging is associated with a number of both storage and voiding problems which are classified into syndromes with overlapping symptoms. Despite the prevalence and consequences of these syndromes, LUT disorders continue to be undertreated as few therapeutic options exist. Here, we propose that dysregulated metabolism of purine nucleotides results in an accumulation of uro-damaging hypoxanthine (a source of reactive oxygen species or ROS), which provides a mechanism for defects in sensory signaling and contractility, culminating in abnormal urodynamic behavior.

Proteomic Analysis of Estrogen-Mediated Enhancement of Mesenchymal Stem Cell-Induced Angiogenesis In Vivo.

Therapeutic use of mesenchymal stem cells (MSCs) for tissue repair has great potential. MSCs from multiple sources, including those derived from human umbilical matrix, namely Wharton's jelly, may serve as a resource for obtaining MSCs. However, low in vivo engraftment efficacy of MSCs remains a challenging limitation. To improve clinical outcomes using MSCs, an in-depth understanding of the mechanisms and factors involved in successful engraftment is required. We recently demonstrated that 17ß-estradiol (E2) improves MSCs in vitro proliferation, directed migration and engraftment in murine heart slices. Here, using a proteomics approach, we investigated the angiogenic potential of MSCs in vivo and the modulatory actions of E2 on mechanisms involved in tissue repair. Specifically, using a Matrigel® plug assay, we evaluated the effects of E2 on MSCs-induced angiogenesis in ovariectomized (OVX) mice. Moreover, using proteomics we investigated the potential pro-repair processes, pathways, and co-mechanisms possibly modified by the treatment of MSCs with E2. Using RT-qPCR, we evaluated mRNA expression of pro-angiogenic molecules, including endoglin, Tie-2, ANG, and VEGF. Hemoglobin levels, a marker for blood vessel formation, were increased in plugs treated with E2 + MSCs, suggesting increased capillary formation. This conclusion was confirmed by the histological analysis of capillary numbers in the Matrigel® plugs treated with E2 + MSC. The LC-MS screening of proteins obtained from the excised Matrigel® plugs revealed 71 proteins that were significantly altered following E2 exposure, 57 up-regulated proteins and 14 down-regulated proteins. A major result was the association of over 100 microRNA molecules (miRNAs) involved in cellular communication, vesicle transport, and metabolic and energy processes, and the high percentage of approximately 25% of genes involved in unknown biological processes. Together, these data provide evidence for increased angiogenesis by MSCs treated with the sex hormone E2. In conclusion, E2 treatment may increase the engraftment and repair potential of MSCs into tissue, and may promote MSC-induced angiogenesis after tissue injury.

The Adenosine Pathway and Human Immunodeficiency Virus-Associated Inflammation.

Human immunodeficiency virus (HIV) is associated with an increased risk of age-associated comorbidities and mortality compared to people without HIV. This has been attributed to HIV-associated chronic inflammation and immune activation despite viral suppression. The adenosine pathway is an established mechanism by which the body regulates persistent inflammation to limit tissue damage associated with inflammatory conditions. However, HIV infection is associated with derangements in the adenosine pathway that limits its ability to control HIV-associated inflammation. This article reviews the function of purinergic signaling and the role of the adenosine signaling pathway in HIV-associated chronic inflammation. This review also discusses the beneficial and potential detrimental effects of pharmacotherapeutic strategies targeting this pathway among people with HIV.

Breast Cancer Cell-Derived Adenosine Enhances Generation and Suppressor Function of Human Adaptive Regulatory T Cells.

INTRODUCTION: Adaptive regulatory T cells (Tr1) are induced in the periphery by environmental stimuli. CD73 expression and adenosine (ADO) production by tumor cells may influence Tr1 generation and their immunosuppressive activity. MATERIAL AND METHODS: Tr1 were generated in co-cultures of CD4+CD25neg T cells, autologous immature dendritic cells (iDC), and irradiated ADO-producing CD73+ or non-producing CD73neg breast cancer (BrCa) cell lines (TU). The expression of ectonucleotidases and other surface markers on Tr1 was determined by flow cytometry. Tr1-mediated suppression of proliferation was evaluated in CFSE-based assays. Luciferase-based ATP detection assays and mass spectrometry were used to measure ATP hydrolysis and ADO levels. Cytokine levels were measured by ELISA or Luminex. CD73 expression on tumor cells or T cells in TU tissues was assessed by immunofluorescence. RESULTS: CD73+ TU induced higher numbers of Tr1 cells (p < 0.01) than CD73neg TU. Tr1TU73+ hydrolyzed more exogenous ATP, produced more ADO, and mediated higher suppression than Tr1TU73neg (p < 0.05 for all). ARL67156, an ectonucleotidase inhibitor, and ZM241385, A2A receptor antagonist, reduced suppression of proliferation mediated by Tr1TU73+ cells (p < 0.01). Basal-like primary BrCa cells expressed higher levels of ectonucleotidases and induced more Tr1 than less aggressive primary luminal-like BrCa. CONCLUSION: BrCa producing ADO (CD73+ TU) favor the induction of Tr1, which expresses CD39 and CD73, hydrolyzes ATP to ADO, and effectively suppresses anti-tumor immunity.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved. This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

KIM-1-mediated anti-inflammatory activity is preserved by MUC1 induction in the proximal tubule during ischemia-reperfusion injury.

Cell-associated kidney injury molecule-1 (KIM-1) exerts an anti-inflammatory role following kidney injury by mediating efferocytosis and downregulating the NF-κB pathway. KIM-1 cleavage blunts its anti-inflammatory activities. We reported that mucin 1 (MUC1) is protective in a mouse model of ischemia-reperfusion injury (IRI). As both KIM-1 and MUC1 are induced in the proximal tubule (PT) during IRI and are a disintegrin and metalloprotease 17 (ADAM17) substrates, we tested the hypothesis that MUC1 protects KIM-1 activity. Muc1 knockout (KO) mice and wild-type (WT) littermates were subjected to IRI. KIM-1, MUC1, and ADAM17 levels (and signaling pathways) were assessed by immunoblot analysis. PT localization was assessed by confocal microscopy and an in situ proximity ligation assay. Findings were extended using human kidneys and urine as well as KIM-1-mediated efferocytosis assays in mouse PT cultures. In response to tubular injury in mouse and human kidneys, we observed induction and coexpression of KIM-1 and MUC1 in the PT. Compared with WT mice, Muc1 KO mice had higher urinary KIM-1 and lower kidney KIM-1. KIM-1 was apical in the PT of WT kidneys but predominately with luminal debris in Muc1 KO mice. Efferocytosis was reduced in Muc1 KO PT cultures compared with WT cultures, whereas inflammation was increased in Muc1 KO kidneys compared with WT kidneys. MUC1 was cleaved by ADAM17 in PT cultures and blocked KIM-1 shedding in Madin-Darby canine kidney cells. We conclude that KIM-1-mediated efferocytosis and thus anti-inflammatory activity during IRI is preserved in the injured kidney by MUC1 inhibition of KIM-1 shedding.NEW & NOTEWORTHY KIM-1 plays a key role in the recovery of the tubule epithelium during renal IRI by mediating efferocytosis and associated signaling that suppresses inflammation. Excessive cleavage of KIM-1 by ADAM17 provides a decoy receptor that aggravates efferocytosis and subsequent signaling. Our data from experiments in mice, patients, and cultured cells show that MUC1 is also induced during IRI and competes with KIM-1 for cleavage by ADAM17. Consequently, MUC1 protects KIM-1 anti-inflammatory activity in the damaged kidney.

Modulation of Cyclic AMP Levels in Fallopian Tube Cells by Natural and Environmental Estrogens.

Autocrine/paracrine factors generated in response to 17ß-estradiol (E2) within the fallopian tube (FT) facilitate fertilization and early embryo development for implantation. Since cyclic AMP (cAMP) plays a key role in reproduction, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Herein, we investigated whether cAMP production in FT cells (FTCs) is regulated by E2 and environmental estrogens (EE's; xenoestrogens and phytoestrogens). Under basal conditions, low levels of extracellular cAMP were detectable in bovine FTCs (epithelial cells and fibroblasts; 1:1 ratio). Treatment of FTCs with forskolin (AC; adenylyl cyclase activator), isoproterenol (ß-adrenoceptor agonist) and IBMX (phosphodiesterase (PDE) inhibitor) dramatically (>10 fold) increased cAMP; whereas LRE1 (sAC; soluble AC inhibitor) and 2',5'-dideoxyadenosine (DDA; transmembrane AC (tmAC)) inhibitor decreased cAMP. Comparable changes in basal and stimulated intracellular cAMP were also observed. Ro-20-1724 (PDE-IV inhibitor), but not milrinone (PDE-III inhibitor) nor mmIBMX (PDE-I inhibitor), augmented forskolin-stimulated cAMP levels, suggesting that PDE-IV dominates in FTCs. E2 increased cAMP levels and CREB phosphorylation in FTCs, and these effects were mimicked by EE's (genistein, 4-hydroxy-2',4',6'-trichlorobiphenyl, 4-hydroxy-2',4',6'-dichlorobiphenyl). Moreover, the effects of E2 and EE were blocked by the tmAC inhibitor DDA, but not by the ERα/ß antagonist ICI182780. Moreover, BAPTA-AM (intracellular-Ca2+ chelator) abrogated the effects of E2, but not genistein, on cAMP suggesting differential involvement of Ca2+. Treatment with non-permeable E2-BSA induced cAMP levels and CREB-phosphorylation; moreover, the stimulatory effects of E2 and EEs on cAMP were blocked by G15, a G protein-coupled estrogen receptor (GPER) antagonist. E2 and IBMX induced cAMP formation was inhibited by LRE1 and DDA suggesting involvement of both tmAC and sAC. Our results provide the first evidence that in FTCs, E2 and EE's stimulate cAMP synthesis via GPER. Exposure of the FT to EE's and PDE inhibitors may result in abnormal non-cyclic induction of cAMP levels which may induce deleterious effects on reproduction.

Long-Term Dipeptidyl Peptidase 4 Inhibition Worsens Hypertension and Renal and Cardiac Abnormalities in Obese Spontaneously Hypertensive Heart Failure Rats.

Background The long-term effects of dipeptidyl peptidase 4 (DPP4) inhibitors on blood pressure and cardiovascular and renal health remain controversial. Herein, we investigated the extended (>182 days) effects of DPP4 inhibition in a model of spontaneous hypertension, heart failure, diabetes mellitus, obesity and hyperlipidemia. Methods and Results Adult obese spontaneously hypertensive heart failure rats (SHHF) were implanted with radio transmitters for measurement of arterial blood pressures. Two weeks later, SHHF were randomized to receive either a DPP4 inhibitor (sitagliptin, 80 mg/kg per day in drinking water) or placebo. At the end of the radiotelemetry measurements, renal and cardiac function and histology, as well as other relevant biochemical parameters, were assessed. For the first 25 days, mean arterial blood pressures were similar in sitagliptin-treated versus control SHHF; afterwards, mean arterial blood pressures increased more in sitagliptin-treated SHHF (P<0.000001). The time-averaged mean arterial blood pressures from day 26 through 182 were 7.2 mm Hg higher in sitagliptin-treated SHHF. Similar changes were observed for systolic (8.6 mm Hg) and diastolic (6.1 mm Hg) blood pressures, and sitagliptin augmented hypertension throughout the light-dark cycle. Long-term sitagliptin treatment also increased kidney weights, renal vascular resistances, the excretion of kidney injury molecule-1 (indicates injury to proximal tubules), renal interstitial fibrosis, glomerulosclerosis, renal vascular hypertrophy, left ventricular dysfunction, right ventricular degeneration, and the ratios of collagen IV/collagen III and collagen IV/laminin in the right ventricle. Conclusions These findings indicate that, in some genetic backgrounds, long-term DPP4 inhibitor treatment is harmful and identify an animal model to study mechanisms of, and test ways to prevent, DPP4 inhibitor-induced pathological conditions.