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Neutrophil extracellular traps (NETs) are associated with rheumatoid arthritis pathogenesis and severity. Since homeostatic NET-forming neutrophils [NET+Ns] have beneficial roles in defense against pathogens, their distinction from pro-injury [NET+N] subtypes is important, especially if they are to be therapeutically-targeted. Having identified circulating, pro-injury DEspR+CD11b+ [NET+Ns] in patients with neutrophilic secondary tissue injury, we determined whether DEspR+ [NET+Ns] are present in RA-flares. Whole blood samples of patients with RA-flares on maintenance therapy (n=6), were analyzed by flow cytometry (FCM) and immunofluorescence cytology followed by semi-automated quantitative confocal microscopy (qIFC). We assessed clinical parameters, levels of neutrophils and [NET+Ns], and plasma S100A8/A9. qIFC detected circulating DEspR+CD11b+ neutrophils and [NET+Ns] in RA-flare patients but not healthy controls. DEspR+ [NET+Ns] were positive for citrullinated histone H3 (citH3+), extruded DNA, decondensed but recognizable polymorphic nuclei, and [NET+N] doublet-interactions in mostly non-ruptured NET-forming neutrophils. Circulating DNA+/DEspR+/CD11b+/citH3+ microvesicles (netMVs) were observed. FCM detected increased %DEspR+CD11b+ neutrophils and DEspR+ cell-cell doublets whose levels trended with DAS28 scores, as did plasma S100A8/A9 levels. This study identifies circulating DEspR+/CD11b+ neutrophils and [NET+Ns] in RA-flare patients on maintenance therapy. Detection of circulating DEspR+citH3+ [NET+Ns] and netMVs indicate a systemic neutrophilic source of citH3-antigen concordant with multi-joint RA pathogenesis. Increased S100A8/A9 alarmin levels are associated with cell injury and released upon NET-formation. As a ligand for TLR4, S100A8/A9 forms a positive feedback loop for TLR4-induced DEspR+ neutrophils. These data identify DEspR+ neutrophils and [NET+Ns] in RA pathogenesis as a potential biomarker and/or therapeutic target.
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BACKGROUND: Pancreatic peritoneal carcinomatosis (PPC), with the worst median overall-survival (mOS), epitomizes the incurability of metastatic cancer. Cancer stem cells (CSCs) underpin this incurability. However, inhibitors of CSC-stemness fail to increase mOS in cancer patients despite preclinical tumor-reduction. This shortfall reinforces that preclinical efficacy should be defined by increased mOS in the presence of cancer comorbidities, CSC-heterogeneity and plasticity. The primary objectives of this study are: to test the dual endothelin-1/signal peptide receptor, DEspR, as a nodal therapeutic target in PPC, given DEspR induction in anoikis-resistant pancreatic CSCs, and to validate humanized anti-DEspR antibody, hu-6g8, as a potential therapeutic for PPC. METHODS: We used heterogeneous pools of CSCs selected for anoikis resistance from reprogrammed Panc1 and MiaPaCa2 tumor cells (TCs), and adherent TCs reprogrammed from CSCs (cscTCs). We used multiple anti-DEspR blocking antibodies (mAbs) with different epitopes, and a humanized anti-DEspR recombinant mAb cross-reactive in rodents and humans, to test DEspR inhibition effects. We measured DEspR-inhibition efficacy on multiple prometastatic CSC-functions in vitro, and on tumorigenesis and overall survival in a CSC-derived xenograft (CDX) nude rat model of PPC with comorbidities. RESULTS: Here we show that DEspR, a stress-survival receptor, is present on subsets of PDAC Panc1-TCs, TC-derived CSCs, and CSC-differentiated TCs (cscTCs), and that DESpR-inhibition decreases apoptosis-resistance and pro-metastatic mesenchymal functions of CSCs and cscTCs in vitro. We resolve the DNA-sequence/protein-function discordance by confirming ADAR1-RNA editing-dependent DEspR-protein expression in Panc1 and MiaPaCa2 TCs. To advance DEspR-inhibition as a nodal therapeutic approach for PPC, we developed and show improved functionality of a recombinant, humanized anti-DEspR IgG4S228P antibody, hu-6g8, over murine precursor anti-DEspR mabs. Hu-6g8 internalizes and translocates to the nucleus colocalized with cyto-nuclear shuttling galectins-1/3, and induces apoptotic cell changes. DEspR-inhibition blocks transperitoneal dissemination and progression to peritoneal carcinomatosis of heterogeneous DEspR±/CD133 ± Panc1-derived CSCs in xenografted nude rats, improving mOS without chemotherapy-like adverse effects. Lastly, we show DEspR expression in Stage II-IV primary and invasive TCs in the stroma in PDAC-patient tumor arrays. CONCLUSION: Collectively, the data support humanized anti-DEspR hu-6g8 as a potential targeted antibody-therapeutic with promising efficacy, safety and prevalence profiles for PPC patients.
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Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos Inmunológicos/farmacología , Inmunoglobulina G/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Neoplasias Peritoneales/tratamiento farmacológico , Neoplasias Peritoneales/secundario , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/química , Antineoplásicos Inmunológicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Inmunoglobulina G/química , Inmunohistoquímica , Inmunofenotipificación , Neoplasias Pancreáticas/patología , Ratas , Receptor de Endotelina A , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: In contrast to rat and mouse databases, the NCBI gene database lists the human dual-endothelin1/VEGFsp receptor (DEspR, formerly Dear) as a unitary transcribed pseudogene due to a stop [TGA]-codon at codon#14 in automated DNA and RNA sequences. However, re-analysis is needed given prior single gene studies detected a tryptophan [TGG]-codon#14 by manual Sanger sequencing, demonstrated DEspR translatability and functionality, and since the demonstration of actual non-translatability through expression studies, the standard-of-excellence for pseudogene designation, has not been performed. Re-analysis must meet UNIPROT criteria for demonstration of a protein's existence at the highest (protein) level, which a priori, would override DNA- or RNA-based deductions. METHODS: To dissect the nucleotide sequence discrepancy, we performed Maxam-Gilbert sequencing and reviewed 727 RNA-seq entries. To comply with the highest level multiple UNIPROT criteria for determining DEspR's existence, we performed various experiments using multiple anti-DEspR monoclonal antibodies (mAbs) targeting distinct DEspR epitopes with one spanning the contested tryptophan [TGG]-codon#14, assessing: (a) DEspR protein expression, (b) predicted full-length protein size, (c) sequence-predicted protein-specific properties beyond codon#14: receptor glycosylation and internalization, (d) protein-partner interactions, and (e) DEspR functionality via DEspR-inhibition effects. RESULTS: Maxam-Gilbert sequencing and some RNA-seq entries demonstrate two guanines, hence a tryptophan [TGG]-codon#14 within a compression site spanning an error-prone compression sequence motif. Western blot analysis using anti-DEspR mAbs targeting distinct DEspR epitopes detect the identical glycosylated 17.5 kDa pull-down protein. Decrease in DEspR-protein size after PNGase-F digest demonstrates post-translational glycosylation, concordant with the consensus-glycosylation site beyond codon#14. Like other small single-transmembrane proteins, mass spectrometry analysis of anti-DEspR mAb pull-down proteins do not detect DEspR, but detect DEspR-protein interactions with proteins implicated in intracellular trafficking and cancer. FACS analyses also detect DEspR-protein in different human cancer stem-like cells (CSCs). DEspR-inhibition studies identify DEspR-roles in CSC survival and growth. Live cell imaging detects fluorescently-labeled anti-DEspR mAb targeted-receptor internalization, concordant with the single internalization-recognition sequence also located beyond codon#14. CONCLUSIONS: Data confirm translatability of DEspR, the full-length DEspR protein beyond codon#14, and elucidate DEspR-specific functionality. Along with detection of the tryptophan [TGG]-codon#14 within an error-prone compression site, cumulative data demonstrating DEspR protein existence fulfill multiple UNIPROT criteria, thus refuting its pseudogene designation.
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Biosíntesis de Proteínas , Seudogenes/genética , Animales , Anoicis , Línea Celular Tumoral , Codón , Galectina 1/análisis , Galectina 1/metabolismo , Humanos , Ratones , Neoplasias/genética , Neoplasias/metabolismo , Mapas de Interacción de Proteínas , Ratas , Triptófano/genéticaRESUMEN
BACKGROUND: Arterial stiffness is an independent predictor of cardiovascular outcomes in hypertensive patients including myocardial infarction, fatal stroke, cerebral micro-bleeds which predicts cerebral hemorrhage in hypertensive patients, as well as progression to hypertension in non-hypertensive subjects. The association between arterial stiffness and various cardiovascular outcomes (coronary heart disease, stroke) remains after adjusting for age, sex, blood pressure, body mass index and other known predictors of cardiovascular disease, suggesting that arterial stiffness, measured via carotid-femoral pulse wave velocity, has a better predictive value than each of these factors. Recent evidence shows that arterial stiffening precedes the onset of high blood pressure; however their molecular genetic relationship (s) and sex-specific determinants remain uncertain. We investigated whether distinct or shared genetic determinants might underlie susceptibility to arterial stiffening in male and female Dahl salt-sensitive rats. Thus, we performed a genome-wide scan for quantitative trait loci (QTLs) affecting arterial stiffness in six-week old F2 (Dahl S x R)-intercross male and female rats characterized for abdominal aortic pulse wave velocity and aortic strain by high-resolution ultrasonography. RESULTS: We detected five highly significant QTLs affecting aortic stiffness: two interacting QTLs (AS-m1 on chromosome 4 and AS-m2 on chromosome16, LOD 8.8) in males and two distinct interacting QTLs (AS-f1 on chromosome 9 and AS-f2 on chromosome11, LOD 8.9) in females affecting pulse wave velocity. One QTL (AS-1 on chromosome 3, LOD 4.3) was found to influence aortic strain in a sex-independent manner. None of these arterial stiffness QTLs co-localized with previously reported blood pressure QTLs detected in equivalent genetic intercrosses. CONCLUSIONS: These data reveal sex-specific genetic determinants for aortic pulse wave velocity and suggest distinct polygenic susceptibility for arterial stiffness and salt-sensitive hypertension in Dahl rats based upon reported blood pressure QTLs in equivalent (Dahl S x R)-intercrosses.
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Hipertensión/genética , Caracteres Sexuales , Rigidez Vascular/genética , Animales , Presión Sanguínea/genética , Cruzamientos Genéticos , Femenino , Genoma , Masculino , Sitios de Carácter Cuantitativo , Ratas , Ratas Endogámicas DahlAsunto(s)
Presión Sanguínea/fisiología , Endotelio Vascular/fisiología , Hipertensión/economía , National Heart, Lung, and Blood Institute (U.S.)/tendencias , Informe de Investigación/tendencias , Rigidez Vascular/fisiología , Endotelio Vascular/citología , Humanos , Hipertensión/epidemiología , Hipertensión/terapia , National Heart, Lung, and Blood Institute (U.S.)/economía , Estados Unidos/epidemiologíaRESUMEN
Background: Cumulative research show association of neutrophils and neutrophil extracellular traps (NETs) with poor outcomes in severe COVID-19. However, to date, there is no curative intent therapy able to block neutrophil/NETs-mediated progression of multi-organ dysfunction. Because of emerging neutrophil heterogeneity, the study of subsets of circulating NET-forming neutrophils [NET + Ns] as mediators of multi-organ failure progression among patients with COVID-19 is critical to identification of therapeutic targets. Methods: We conducted a prospective observational study of circulating levels of CD11b + [NET + N] immunotyped for dual endothelin-1/signal peptide receptor (DEspR ±) expression by quantitative immunofluorescence-cytology and causal mediation analysis. In 36 consented adults hospitalized with mod-severe COVID-19, May to September 2020, we measured acute multi-organ failure via SOFA-scores and respiratory failure via SaO2/FiO2 (SF)-ratio at time points t1 (average 5.5 days from ICU/hospital admission) and t2 (the day before ICU-discharge or death), and ICU-free days at day28 (ICUFD). Circulating absolute neutrophil counts (ANC) and [NET + N] subset-specific counts were measured at t1. Spearman correlation and causal mediation analyses were conducted. Results: Spearman correlation analyses showed correlations of t1-SOFA with t2-SOFA (rho r S = 0.80) and ICUFD (r S = -0.76); circulating DEspR + [NET + Ns] with t1-SOFA (r S = 0.71), t2-SOFA (r S = 0.62), and ICUFD (r S = -0.63), and ANC with t1-SOFA (r S = 0.71), and t2-SOFA (r S = 0.61).Causal mediation analysis identified DEspR + [NET + Ns] as mediator of 44.1% [95% CI:16.5,110.6] of the causal path between t1-SOFA (exposure) and t2-SOFA (outcome), with 46.9% [15.8,124.6] eliminated when DEspR + [NET + Ns] were theoretically reduced to zero. Concordantly, DEspR + [NET + Ns] mediated 47.1% [22.0,72.3%] of the t1-SOFA to ICUFD causal path, with 51.1% [22.8,80.4%] eliminated if DEspR + [NET + Ns] were reduced to zero. In patients with t1-SOFA > 1, the indirect effect of a hypothetical treatment eliminating DEspR + [NET + Ns] projected a reduction of t2-SOFA by 0.98 [0.29,2.06] points and ICUFD by 3.0 [0.85,7.09] days. In contrast, there was no significant mediation of SF-ratio through DEspR + [NET + Ns], and no significant mediation of SOFA-score through ANC. Conclusions: Despite equivalent correlations, DEspR + [NET + Ns], but not ANC, mediated progression of multi-organ failure in acute COVID-19, and its hypothetical reduction is projected to improve ICUFD. These translational findings warrant further studies of DEspR + [NET + Ns] as potential patient-stratifier and actionable therapeutic target for multi-organ failure in COVID-19. Supplementary Information: The online version contains supplementary material available at 10.1186/s41231-023-00143-x.
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Background: Cumulative research show association of neutrophils and neutrophil extracellular traps (NETs) with poor outcomes in severe COVID-19. However, to date, no curative intent therapy has been identified to block neutrophil/NETs-mediated progression of multi-organ dysfunction. Because of emerging neutrophil heterogeneity, the study of subsets of circulating neutrophil-extracellular trap (NET)-forming neutrophils [NET+Ns] as mediators of multi-organ failure progression among patients with COVID-19 is critical to identification of therapeutic targets. Methods: We conducted a prospective observational study of circulating levels of CD11b+[NET+N] immunotyped for dual endothelin-1/signal peptide receptor, (DEspR±) expression by quantitative immunofluorescence-cytology and causal mediation analysis. In 36 consented adults hospitalized with mod-severe COVID-19, May to September 2020, we measured acute multi-organ failure via SOFA-scores and respiratory failure via SaO2/FiO2 (SF)ratio at time points t1 (average 5.5 days from ICU/hospital admission) and t2 (the day before ICU-discharge or death), and ICU-free days at day28 (ICUFD). Circulating absolute neutrophil counts (ANC) and [NET+N] subset-specific counts were measured at t1. Spearman correlation and causal mediation analyses were conducted. Results: Spearman correlation analyses showed correlations of t1-SOFA with t2-SOFA ( rho r S =0.80) and ICUFD ( r S =-0.76); circulating DEspR+[NET+Ns] with t1-SOFA ( r S = 0.71), t2-SOFA ( r S =0.62), and ICUFD ( r S =-0.63), and ANC with t1-SOFA ( r S =0.71), and t2-SOFA ( r S =0.61). Causal mediation analysis identified DEspR+[NET+Ns] as mediator of 44.1% [95% CI:16.5,110.6] of the causal path between t1-SOFA (exposure) and t2-SOFA (outcome), with 46.9% [15.8,124.6] eliminated when DEspR+[NET+Ns] were theoretically reduced to zero. Concordantly, DEspR+[NET+Ns] mediated 47.1% [22.0,72.3%] of the t1-SOFA to ICUFD causal path, with 51.1% [22.8,80.4%] eliminated if DEspR+[NET+Ns] were reduced to zero. In patients with t1-SOFA >1, the indirect effect of a hypothetical treatment eliminating DEspR+[NET+Ns] projected a reduction of t2-SOFA by 0.98 [0.29,2.06] points and ICUFD by 3.0 [0.85,7.09] days. In contrast, there was no significant mediation of SF-ratio through DEspR+[NET+Ns], and no significant mediation of SOFA-score through ANC. Conclusions: Despite equivalent correlations, DEspR+[NET+Ns], but not ANC, mediated progression of multi-organ failure in acute COVID-19, and its hypothetical reduction is projected to improve ICUFD. These translational findings warrant further studies of DEspR+[NET+Ns] as potential patient-stratifier and actionable therapeutic target for multi-organ failure in COVID-19.
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Progressive secondary brain injury-induced by dysregulated neuroinflammation in spontaneous intracerebral hemorrhage (sICH)-underlies high sICH-mortality and remains without FDA-approved pharmacotherapy. Clinical insight that hematoma-directed interventions do not improve mortality prioritizes resolving acute secondary brain injury in sICH. As neutrophils are implicated in sICH secondary brain injury, we tested whether inhibition of a rogue neutrophil-subset expressing the dual endothelin-1/signal peptide receptor (DEspR) and associated with secondary tissue injury, DEspR+ CD11b+ immunotype, will attenuate mortality in a hypertensive-sICH (hsICH) rat model. We confirmed sICH-related deaths in hsICH-rats by T2*-weighted 9.4 T MRI and DEspR+ neutrophils in hsICH-rat brain perihematomal areas by immunostaining. At acute sICH, anti-DEspR muIgG1-antibody, mu10a3, treatment increased median survival in hsICH rats vs controls (p < 0.0001). In pre-stroke sICH, weekly 10a3-treatment did not predispose to infection and delayed sICH-onset vs controls (p < 0.0001). As potential sICH-therapeutic, we tested humanized anti-DEspR IgG4S228P-mAb, hu6g8. In vitro, hu6g8 reversed delayed-apoptosis in DEspR+ CD11b+ neutrophils. In vivo, hu6g8 increased median survival and reduced neurologic symptoms in male/female hsICH-rats vs controls (p < 0.0001). Altogether, preclinical efficacy of inhibition of DEspR+ CD11b+ neutrophils in acute sICH-without infection complications, supports the potential of anti-DEspR therapy in sICH. Data provide basis for clinical study of DEspR+ CD11b+ neutrophil-subset in sICH patients.
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Lesiones Encefálicas , Hipertensión , Accidente Cerebrovascular , Animales , Femenino , Masculino , Ratas , Lesiones Encefálicas/complicaciones , Hemorragia Cerebral/complicaciones , Hemorragia Cerebral/tratamiento farmacológico , Hipertensión/complicaciones , Accidente Cerebrovascular/complicaciones , SeudogenesRESUMEN
Background and objective: The correlation (Rs > 0.7) of neutrophils expressing the dual endothelin1/signal peptide receptor (DEspR+CD11b+/CD66b+) with severity of hypoxemia (SF-ratio) and multi-organ failure (SOFA-score) in patients with acute respiratory distress syndrome (ARDS) suggest the hypothesis that the DEspR+ neutrophil-subset is an actionable therapeutic target in ARDS. To test this hypothesis, we conducted in vivo studies to validate DEspR+ neutrophil-subset as therapeutic target and test efficacy of DEspR-inhibition in acute neutrophilic hyperinflammation models. Methods: We performed tests in lipopolysaccharide (LPS)-induced acute neutrophilic inflammation in three species - human, rhesus macaque, rat - with increasing dose-dependent severity. We measured DEspR+CD66b+ neutrophils in bronchoalveolar lavage fluid (BALF) in healthy volunteers (HVs) 24-hours after segmental LPS-challenge by ChipCytometry, and DEspR+CD11b+ neutrophils in whole blood and BALF in an LPS-induced transient acute lung injury (ALI) model in macaques. We determined anti-DEspR antibody efficacy in vivo in LPS-ALI macaque model and in high-mortality LPS-induced encephalopathy in hypertensive rats. Results: ChipCytometry detected increased BALF total neutrophil and DEspR+CD66b+ neutrophil counts after segmental LPS-challenge compared to baseline (P =0.034), as well as increased peripheral neutrophil counts and neutrophil-lymphocyte ratio (NLR) compared to pre-LPS level (P <0.05). In the LPS-ALI macaque model, flow cytometry detected increased DEspR+ and DEspR[-] neutrophils in BALF, which was associated with moderate-severe hypoxemia. After determining pharmacokinetics of single-dose anti-DEspR[hu6g8] antibody, one-time pre-LPS anti-DEspR treatment reduced hypoxemia (P =0.03) and neutrophil influx into BALF (P =0.0001) in LPS-ALI vs vehicle mock-treated LPS-ALI macaques. Ex vivo live cell imaging of macaque neutrophils detected greater "intrinsic adhesion to hard-surface" in DEspR+ vs DEspR[-] neutrophils (P <0.001). Anti-DEspR[hu6g8] antibody abrogated intrinsic high adhesion in DEspR+ neutrophils, but not in DEspR[-] neutrophils (P <0.001). In the LPS-encephalopathy rat model, anti-DEspR[10a3] antibody treatment increased median survival (P =0.0007) and exhibited brain target engagement and bioeffects. Conclusion: Detection of increased DEspR+ neutrophil-subset in human BALF after segmental LPS-challenge supports the correlation of circulating DEspR+ neutrophil counts with severity measure (SOFA-score) in ARDS. Efficacy and safety of targeted inhibition of DEspR+CD11b+ neutrophil-subset in LPS-induced transient-ALI and high-mortality encephalopathy models identify a potential therapeutic target for neutrophil-mediated secondary tissue injury.
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Lesión Pulmonar Aguda , Encefalopatías , Síndrome de Dificultad Respiratoria , Humanos , Ratas , Animales , Lipopolisacáridos/efectos adversos , Neutrófilos , Macaca mulatta , Lesión Pulmonar Aguda/metabolismo , Inflamación/metabolismo , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Hipoxia/metabolismo , Encefalopatías/metabolismoRESUMEN
Objective: Cumulative clinical, cellular, and molecular evidence reinforces the role of neutrophils in secondary brain injury in spontaneous intracerebral hemorrhage (sICH). However, since generalized neutrophil inhibition is detrimental, identification of targetable "rogue" neutrophil subsets associated with sICH severity is key. Methods: In a pilot prospective observational study of consented patients with sICH, we immunotyped whole blood to assess circulating neutrophil markers (~day 3 after ICH symptoms onset): (a) DEspR±CD11b± neutrophils by flow cytometry, (b) DEspR±CD11b± neutrophil extracellular trap (NET)-forming neutrophils by immunofluorescence cytology, and (c) neutrophil-lymphocyte ratio (NLR). Using Spearman rank correlation (r) with Bonferroni correction, we assessed the association of neutrophil markers with same-day clinical and neuroimaging parameters of sICH severity, index ICH score, 90-day modified Rankin Scale (mRS) score, and potential interrelationships. As comparators, we assessed same-day plasma biomarkers elevated in sICH: interleukin-6/IL-6, myeloperoxidase/MPO, soluble-terminal complement complex/sC5b-9, endothelin-1/ET-1, and mitochondrial/nuclear DNA ratio (mt/nDNA ratio). Results: We detected strong correlations [r(n = 13) > 0.71, power > 0.8, Bonferroni corrected p B < 0.05] for all three neutrophil markers with 90-day mRS score, differentially for DEspR+CD11b+ neutrophil counts, and NLR with perihematomal edema (PHE) volume and for DEspR+CD11b+ NET-forming neutrophil counts with intraparenchymal hemorrhage (IPH)-volume. Only DEspR+CD11b+ neutrophil counts show a strong correlation with index ICH score, same-day Glasgow Coma Scale (GCS) score, and NLR and NET-forming neutrophil counts. The sum of the ICH score and three neutrophil markers exhibited the highest correlation: [r(n = 13) 0.94, p B = 10-5]. In contrast, plasma biomarkers tested were elevated except for MPO but exhibited no correlations in this pilot study. Conclusion: Strong correlation with multiple sICH severity measures, NET formation, and NLR identifies DEspR+CD11b+ neutrophils as a putative "rogue" neutrophil subset in sICH. The even stronger correlation of the sum of three neutrophil markers and the index ICH score with 90-day mRS outcome reinforces early neutrophil-mediated secondary brain injury as a key determinant of outcome in patients with sICH. Altogether, data provide a basis for the formal study of the DEspR+CD11b+ neutrophil subset as a potential actionable biomarker for neutrophil-driven secondary brain injury in sICH. Data also show ex vivo analysis of patients with sICH neutrophils as a translational milestone to refine hypotheses between preclinical and clinical studies.
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Rationale: High mortality in pancreatic cancer (PDAC) and triple negative breast cancer (TNBC) highlight the need to capitalize on nanoscale-design advantages for multifunctional diagnostics and therapies. DNA/RNA-therapies can provide potential breakthroughs, however, to date, there is no FDA-approved systemic delivery system to solid tumors. Methods: Here, we report a Janus-nanoparticle (jNP)-system with modular targeting, payload-delivery, and targeted-imaging capabilities. Our jNP-system consists of 10 nm ultrasmall superparamagnetic iron oxide nanoparticles (USPION) with opposing antibody-targeting and DNA/RNA payload-protecting faces, directionally self-assembled with commercially available zwitterionic microbubbles (MBs) and DNA/RNA payloads. Results: Sonoporation of targeted jNP-payload-MBs delivers functional reporter-DNA imparting tumor-fluorescence, and micro-RNA126 reducing non-druggable KRAS in PDAC-Panc1 and TNBC-MB231 xenografted tumors. The targeting jNP-system enhances ultrasound-imaging of intra-tumoral microvasculature using less MBs/body weight (BW). The jNP-design enhances USPION's T2*-magnetic resonance (MR) and MR-imaging of PDAC-peritoneal metastases using less Fe/BW. Conclusion: Altogether, data advance the asymmetric jNP-design as a potential theranostic Janus-USPION Modular Platform - a JUMP forward.
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MicroARNs , Neoplasias de la Mama Triple Negativas , Humanos , Medicina de Precisión , Neoplasias de la Mama Triple Negativas/diagnóstico por imagen , Neoplasias de la Mama Triple Negativas/terapia , Diagnóstico por Imagen , ADN , Neoplasias PancreáticasRESUMEN
Neutrophil-mediated secondary tissue injury underlies acute respiratory distress syndrome (ARDS) and progression to multi-organ-failure (MOF) and death, processes linked to COVID-19-ARDS. This secondary tissue injury arises from dysregulated neutrophils and neutrophil extracellular traps (NETs) intended to kill pathogens, but instead cause cell-injury. Insufficiency of pleiotropic therapeutic approaches delineate the need for inhibitors of dysregulated neutrophil-subset(s) that induce subset-specific apoptosis critical for neutrophil function-shutdown. We hypothesized that neutrophils expressing the pro-survival dual endothelin-1/VEGF-signal peptide receptor, DEspR, are apoptosis-resistant like DEspR+ cancer-cells, hence comprise a consequential pathogenic neutrophil-subset in ARDS and COVID-19-ARDS. Here, we report the significant association of increased peripheral DEspR+CD11b+ neutrophil-counts with severity and mortality in ARDS and COVID-19-ARDS, and intravascular NET-formation, in contrast to DEspR[-] neutrophils. We detect DEspR+ neutrophils and monocytes in lung tissue patients in ARDS and COVID-19-ARDS, and increased neutrophil RNA-levels of DEspR ligands and modulators in COVID-19-ARDS scRNA-seq data-files. Unlike DEspR[-] neutrophils, DEspR+CD11b+ neutrophils exhibit delayed apoptosis, which is blocked by humanized anti-DEspR-IgG4S228P antibody, hu6g8, in ex vivo assays. Ex vivo live-cell imaging of Rhesus-derived DEspR+CD11b+ neutrophils showed hu6g8 target-engagement, internalization, and induction of apoptosis. Altogether, data identify DEspR+CD11b+ neutrophils as a targetable 'rogue' neutrophil-subset associated with severity and mortality in ARDS and COVID-19-ARDS.
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COVID-19 , Trampas Extracelulares , Síndrome de Dificultad Respiratoria , Humanos , Inmunofenotipificación , NeutrófilosRESUMEN
Arginine vasopressin (AVP) and angiotensin II (ANG II) are distinct peptide hormones involved in multiple organs modulating renal, cardiovascular, and brain functions. They achieve these functions via specific G protein-coupled receptors, respectively. The AVR/NAVR locus encodes two overlapping V2-type vasopressin isoreceptors: angiotensin-vasopressin receptor (AVR) responding to ANG II and AVP equivalently, and nonangiotensin vasopressin receptor (NAVR), which binds vasopressin exclusively. AVR and NAVR are expressed from a single gene by alternative promoter usage that is synergistically upregulated by testosterone and estrogen. This study tested the hypothesis that AVR/NAVR modulates urinary concentrating ability, blood pressure, and cognitive performance in vivo in a sex-specific manner. We developed a C57BL/6 inbred AVR/NAVR(-/-) knockout mouse that showed lower blood pressure in both male and female subjects and a urinary-concentrating defect restricted to male mice. We also detected sex-specific effects on cognitive and anxiety-like behaviors. AVR/NAVR(-/-) male mice exhibited impaired visuospatial and associative learning, while female mice showed improved performance in both type of cognition. AVR/NAVR deficiency produced an anxiolytic-like effect in female mice, while males were unaffected. Analysis of AVR- and NAVR-mediated phosphorylation/dephosphorylation of signaling proteins revealed activation/deactivation of known modulators of cognitive function. Our studies identify AVR/NAVR as key receptors involved in blood pressure regulation and sex-specific modulation of renal water homeostasis, cognitive function, and anxiety-like behavior. As such, the AVR/NAVR receptor system provides a molecular mechanism for sexually diergic traits and a putative common pathway for the emerging association of hypertension and cognitive decline and dementia.
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Ansiedad/fisiopatología , Presión Sanguínea/fisiología , Cognición/fisiología , Capacidad de Concentración Renal/fisiología , Receptores de Angiotensina/deficiencia , Receptores Acoplados a Proteínas G/deficiencia , Receptores de Vasopresinas/deficiencia , Animales , Ansiedad/genética , Presión Sanguínea/genética , Femenino , Capacidad de Concentración Renal/genética , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Fosforilación , Receptores de Angiotensina/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Vasopresinas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Essential hypertension is highly prevalent in the elderly population, exceeding 70% in people older than 60 yr of age, and remains a leading risk factor for heart disease, stroke, and chronic renal disease. Elucidation of genetic determinants is critical but remains a challenge due to its complex, multifactorial pathogenesis. We investigated the role DEspR promoter variants, previously associated with male essential hypertension susceptibility, in blood pressure (BP) regulation. We detected a single nucleotide polymorphism within the DEspR 5'-regulatory region associated with increased BP in a male Sardinian cohort accounting for 11.0 mmHg of systolic BP (P<10(-15)) and 9.3 mmHg of diastolic BP (P<10(-15)). Sequence analysis of three normotensive subjects homozygous for the rs6535847 "normotension-associated T-allele" identified a canonical TATAAAA-box in contrast to a CATAAAA-motif in three hypertensive subjects homozygous for the rs6535847 "hypertension-associated C-allele." In vitro analysis detected decreased transcription activity with the CATAAAA-motif promoter-construct compared with the canonical TATAAAA-box promoter-construct. Although BP did not differ between DEspR+/- knockout male mice and wild-type littermates at 6 mo of age, radiotelemetric BP measurements in 18 mo old inbred DEspR+/- knockout male mice known to have decreased DEspR RNA and protein detected higher systolic, mean, and diastolic BPs in DEspR+/- mice compared with littermate wild-type controls (P<0.05). Our results demonstrate that promoter variants in DEspR associated with hypertension susceptibility and increased BP in Sardinian males affect transcription levels, which then affect BP in an age-dependent and male-specific manner. This finding is concordant with the late-onset and sex-specific characteristics of essential hypertension, thus reiterating the mandate for sex-specific analyses and treatment approaches for essential hypertension.
Asunto(s)
Presión Sanguínea/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Receptores de Superficie Celular/genética , Transcripción Genética , Anciano , Animales , Secuencia de Bases , Estudios de Asociación Genética , Frecuencia Cardíaca/genética , Heterocigoto , Humanos , Italia , Masculino , Ratones , Datos de Secuencia Molecular , Motivos de Nucleótidos/genética , Reacción en Cadena de la Polimerasa , Seudogenes , Carácter Cuantitativo Heredable , Sitio de Iniciación de la TranscripciónRESUMEN
Stroke is the third leading cause of death in the United States with high rates of morbidity among survivors. The search to fill the unequivocal need for new therapeutic approaches would benefit from unbiased proteomic analyses of animal models of spontaneous stroke in the prestroke stage. Since brain microvessels play key roles in neurovascular coupling, we investigated prestroke microvascular proteome changes. Proteomic analysis of cerebral cortical microvessels (cMVs) was done by tandem mass spectrometry comparing two prestroke time points. Metaprotein-pathway analyses of proteomic spectral count data were done to identify risk factor-induced changes, followed by QSPEC-analyses of individual protein changes associated with increased stroke susceptibility. We report 26 cMV proteome profiles from male and female stroke-prone and non-stroke-prone rats at 2 months and 4.5 months of age prior to overt stroke events. We identified 1,934 proteins by two or more peptides. Metaprotein pathway analysis detected age-associated changes in energy metabolism and cell-to-microenvironment interactions, as well as sex-specific changes in energy metabolism and endothelial leukocyte transmigration pathways. Stroke susceptibility was associated independently with multiple protein changes associated with ischemia, angiogenesis or involved in blood brain barrier (BBB) integrity. Immunohistochemical analysis confirmed aquaporin-4 and laminin-α1 induction in cMVs, representative of proteomic changes with >65 Bayes factor (BF), associated with stroke susceptibility. Altogether, proteomic analysis demonstrates significant molecular changes in ischemic cerebral microvasculature in the prestroke stage, which could contribute to the observed model phenotype of microhemorrhages and postischemic hemorrhagic transformation. These pathways comprise putative targets for translational research of much needed novel diagnostic and therapeutic approaches for stroke.
Asunto(s)
Corteza Cerebral/irrigación sanguínea , Microvasos/metabolismo , Proteoma/análisis , Proteómica/métodos , Animales , Acuaporina 4/análisis , Corteza Cerebral/metabolismo , Circulación Cerebrovascular , Proteínas de Transferencia de Ésteres de Colesterol/genética , Femenino , Humanos , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Inmunohistoquímica , Isquemia/complicaciones , Laminina/análisis , Masculino , Ratas , Ratas Endogámicas Dahl , Ratas Transgénicas , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
SARS-CoV-2 infection results in a spectrum of outcomes from no symptoms to widely varying degrees of illness to death. A better understanding of the immune response to SARS-CoV-2 infection and subsequent, often excessive, inflammation may inform treatment decisions and reveal opportunities for therapy. We studied immune cell subpopulations and their associations with clinical parameters in a cohort of 26 patients with COVID-19. Following informed consent, we collected blood samples from hospitalized patients with COVID-19 within 72 h of admission. Flow cytometry was used to analyze white blood cell subpopulations. Plasma levels of cytokines and chemokines were measured using ELISA. Neutrophils undergoing neutrophil extracellular traps (NET) formation were evaluated in blood smears. We examined the immunophenotype of patients with COVID-19 in comparison to that of SARS-CoV-2 negative controls. A novel subset of pro-inflammatory neutrophils expressing a high level of dual endothelin-1 and VEGF signal peptide-activated receptor (DEspR) at the cell surface was found to be associated with elevated circulating CCL23, increased NETosis, and critical-severity COVID-19 illness. The potential to target this subpopulation of neutrophils to reduce secondary tissue damage caused by SARS-CoV-2 infection warrants further investigation.
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COVID-19/inmunología , Neutrófilos/inmunología , Seudogenes/inmunología , Anciano , Quimiocinas/metabolismo , Estudios de Cohortes , Enfermedad Crítica , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Trampas Extracelulares/metabolismo , Femenino , Humanos , Inflamación/metabolismo , Masculino , Persona de Mediana Edad , Neutrófilos/metabolismo , Seudogenes/genética , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Early-life risk factor exposure increases aortic atherosclerosis and blood pressure in humans and animal models; however, limited insight has been gained as to end-organ complications. METHODS AND RESULTS: We investigated the effects of early-life Na exposure (0.23% versus 0.4% NaCl regular rat chow) on vascular disease outcomes using the inbred, transgenic [hCETP](25) Dahl salt-sensitive hypertensive rat model of male-predominant coronary atherosclerosis, Tg25. Rather than the expected increase in coronary heart disease, fetal 0.4% Na exposure (< or =2 g of Na per 2-kcal/d diet) induced adult-onset stroke in both sexes (ANOVA P<0.0001), with earlier stroke onset in Tg25 females. Analysis of later onset of 0.4% Na exposure resulted in decreased stroke risk and later stroke onset despite longer 0.4% Na exposure durations, which indicates increasing risk with earlier onset of 0.4% Na exposure. Histological analysis of stroke-positive rat brains revealed cerebral cortical hemorrhagic infarctions, microhemorrhages, neuronal ischemia, and microvascular injury. Ex vivo MRI of stroke-positive rat brains detected cerebral hemorrhages, microhemorrhages, and ischemia with middle cerebral artery distribution and cerebellar noninvolvement. Ultrasound microimaging detected carotid artery disease. Prestroke analysis detected neuronal ischemia and decreased mass of isolated cerebral but not cerebellar microvessels. CONCLUSIONS: Early-life Na exposure exacerbated hypertension and unmasked stroke susceptibility, with greater female vulnerability in hypertensive, hyperlipidemic Tg25 rats. The reproducible modeling in stroke-prone Tg25 rats of carotid artery disease, cerebral hemorrhagic infarctions, neuronal ischemia, microhemorrhages, and microvascular alterations suggests a pathogenic spectrum with causal interrelationships. This "mixed-stroke" spectrum could represent paradigms of ischemic-hemorrhagic transformation and/or a microangiopathic basis for the association of ischemic lesions, microhemorrhages, and strokes in humans. Together, the data reveal early-life Na exposure to be a significant modifier of hypertension and stroke disease course and hence a potentially modifiable prevention target that deserves systematic study.
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Proteínas de Transferencia de Ésteres de Colesterol/genética , Hiperlipidemias/genética , Hipertensión/genética , Cloruro de Sodio Dietético/farmacología , Accidente Cerebrovascular/genética , Factores de Edad , Animales , Encéfalo/irrigación sanguínea , Encéfalo/patología , Enfermedades de las Arterias Carótidas/diagnóstico por imagen , Modelos Animales de Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Hiperlipidemias/complicaciones , Hipertensión/complicaciones , Imagen por Resonancia Magnética , Masculino , Microcirculación , Fenotipo , Ratas , Ratas Endogámicas Dahl , Ratas Transgénicas , Factores Sexuales , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/patología , UltrasonografíaRESUMEN
Essential hypertension remains a major risk factor for cardiovascular and cerebrovascular diseases. As a complex multifactorial disease, elucidation of susceptibility loci remains elusive. ATP1A1 and Dear are candidate genes for 2 closely linked rat chromosome-2 blood pressure quantitative trait loci. Because corresponding human syntenic regions are on different chromosomes, investigation of ATP1A1 (chromosome [chr]-1p21) and Dear (chr-4q31.3) facilitates genetic analyses of each blood pressure quantitative trait locus in human hypertension. Here we report the association of human ATP1A1 (P<0.000005) and Dear (P<0.03) with hypertension in a relatively isolated, case/control hypertension cohort from northern Sardinia by single-nucleotide polymorphism haplotype analysis. Sex-specific haplotype analyses detected stronger association of both loci with hypertension in males than in females. Haplotype trend-regression analyses support ATP1A1 and Dear as independent susceptibility loci and reveal haplotype-specific association with hypertension and normotension, thus delineating haplotype-specific subsets of hypertension. Although investigation in other cohorts needs to be performed to determine genetic effects in other populations, haplotype subtyping already allows systematic stratification of susceptibility and, hence, clinical heterogeneity, a prerequisite for unraveling the polygenic etiology and polygene-environment interactions in essential hypertension. As hypertension susceptibility genes, coexpression of ATP1A1 and Dear in both renal tubular cells and vascular endothelium suggest a cellular pathogenic scaffold for polygenic mechanisms of hypertension, as well as the hypothesis that ATP1A1 and/or Dear could contribute to the known renal and vascular endothelial dysfunction associated with essential (polygenic) hypertension.
Asunto(s)
Haplotipos , Hipertensión/genética , Polimorfismo de Nucleótido Simple , Receptores de Angiotensina/genética , Receptores de Endotelina/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Frecuencia de los Genes , Humanos , Desequilibrio de Ligamiento , Análisis de Regresión , Caracteres SexualesRESUMEN
The angiotensin-vasopressin receptor (AVR) responds with equivalent affinities to angiotensin II (ANG II) and vasopressin and is coupled to adenylate cyclase and hence a V2-type vasopressin receptor. AVR maps to the Nalp6 locus and overlaps with the larger Nalp6/PYPAF5 reported to be a T cell/granulocyte-specific, cytoplasmic-specific proapoptotic protein, thus questioning the existence of AVR. Here we confirm, through different experimental modalities, that AVR is distinct from Nalp6/PYPAF5 based on different mRNA and protein sizes, subcellular localization, and tissue-specific expression patterns. Binding studies of PYPAF5-specific Cos1 transfectants detect high-affinity binding to vasopressin but not ANG II, thus assigning PYPAF5 as a non-AVR (NAVR). Signaling array analysis reveals that AVP stimulation of AVR- and NAVR-specific Cos1 transfectants results in diametrical activation as well as coactivation of signaling pathways known to mediate renal sodium and water balance. Likewise, ANG II stimulation of Cos1-AVR transfectants reveals a signaling profile distinct from that of AVP-stimulated Cos1-AVR transfectants. Analysis of genomic organization of the AVR/NAVR locus shows an overlapping gene arrangement with alternative promoter usage resulting in different NH(2) termini for NAVR and AVR. In addition to core promoter elements, androgen and estrogen response elements are detected. Promoter analysis of NAVR/AVR 5'-regulatory region detects transcriptional upregulation by testosterone and synergistic upregulation by testosterone and estrogen, thus suggesting that AVR and/or NAVR contribute to sex-specific V2-type vasopressin-mediated effects. Altogether, confirmation of AVR and identification of NAVR as vasopressin receptors are concordant with emerging vasopressin functions not attributable to V1a, V1b, or V2 receptors and add molecular bases for the multifunctional complexity of vasopressin-mediated functions and regulation.
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Receptores de Angiotensina/genética , Receptores de Vasopresinas/genética , Angiotensina II/metabolismo , Animales , Western Blotting , Células COS , Chlorocebus aethiops , Regulación de la Expresión Génica/efectos de los fármacos , Genoma/genética , Hormonas Esteroides Gonadales/farmacología , Ligandos , Microscopía Confocal , Especificidad de Órganos/efectos de los fármacos , Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores de Angiotensina/metabolismo , Receptores de Vasopresinas/metabolismo , Transducción de Señal/efectos de los fármacos , Sitio de Iniciación de la Transcripción , Transcripción Genética/efectos de los fármacos , Transfección , Vasopresinas/metabolismoRESUMEN
Aside from abnormal angiogenesis, dual endothelin-1/VEGF signal peptide-activated receptor deficiency (DEspR(-/-)) results in aberrant neuroepithelium and neural tube differentiation, thus elucidating DEspR's role in neurogenesis. With the emerging importance of neurogenesis in adulthood, we tested the hypothesis that nonembryonic-lethal DEspR haploinsufficiency (DEspR(+/-)) perturbs neuronal homeostasis, thereby facilitating aging-associated neurodegeneration. Here we show that, in male mice only, DEspR-haploinsufficiency impaired hippocampus-dependent visuospatial and associative learning and induced noninflammatory spongiform changes, neuronal vacuolation, and loss in the hippocampus, cerebral cortex, and subcortical regions, consistent with autophagic cell death. In contrast, DEspR(+/-) females exhibited better cognitive performance than wild-type females and showed absence of neuropathological changes. Signaling pathway analysis revealed DEspR-mediated phosphorylation of activators of autophagy inhibitor mammalian target of rapamycin (mTOR) and dephosphorylation of known autophagy inducers. Altogether, the data demonstrate DEspR-mediated diametrical, sex-specific modulation of cognitive performance and autophagy, highlight cerebral neuronal vulnerability to autophagic dysregulation, and causally link DEspR haploinsufficiency with increased neuronal autophagy, spongiosis, and cognitive decline in mice.