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Novel Pharmaceutical Strategy for Selective Abrogation of TSP1-Induced Vascular Dysfunction by Decoy Recombinant CD47 Soluble Receptor in Prophylaxis and Treatment Models.

ABSTRACT: Elevated thrombospondin 1 (TSP1) is a prevalent factor, via cognate receptor CD47, in the pathogenesis of cardiovascular conditions, including ischemia-reperfusion injury (IRI) and pulmonary arterial hypertension (PAH). Moreover, TSP1/CD47 interaction has been found to be associated with platelet hyperaggregability and impaired nitric oxide response, exacerbating progression in IRI and PAH. Pathological TSP1 in circulation arises as a target of our novel therapeutic approach. Our "proof-of-concept" pharmacological strategy relies on recombinant human CD47 peptide (rh-CD47p) as a decoy receptor protein (DRP) to specifically bind TSP1 and neutralize TSP1-impaired vasorelaxation, strongly implicated in IRI and PAH. The binding of rh-CD47p and TSP1 was first verified as the primary mechanism via Western blotting and further quantified with modified ELISA, which also revealed a linear molar dose-dependent interaction. Ex vivo, pretreatment protocol with rh-CD47p (rh-CD47p added prior to TSP1 incubation) demonstrated a prophylactic effect against TSP1-impairment of endothelium-dependent vasodilation. Post-treatment set-up (TSP1 incubation prior to rh-CD47p addition), mimicking pre-existing excessive TSP1 in PAH, reversed TSP1-inhibited vasodilation back to control level. Dose titration identified an effective molar dose range (approx. ≥1:3 of tTSP1:rh-CD47p) for prevention of/recovery from TSP1-induced vascular dysfunction. Our results indicate the great potential for proposed novel decoy rh-CD47p-therapy to abrogate TSP1-associated cardiovascular complications, such as PAH.

SUBMITTER: Yao M

PROVIDER: S-EPMC8228143 | biostudies-literature |

REPOSITORIES: biostudies-literature

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TSP1-CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction.

Project description:AimsThrombospondin-1 (TSP1) is a ligand for CD47 and TSP1-/- mice are protected from pulmonary hypertension (PH). We hypothesized the TSP1-CD47 axis is upregulated in human PH and promotes pulmonary arterial vasculopathy.Methods and resultsWe analyzed the molecular signature and functional response of lung tissue and distal pulmonary arteries (PAs) from individuals with (n = 23) and without (n = 16) PH. Compared with controls, lungs and distal PAs from PH patients showed induction of TSP1-CD47 and endothelin-1/endothelin A receptor (ET-1/ETA) protein and mRNA. In control PAs, treatment with exogenous TSP1 inhibited vasodilation and potentiated vasoconstriction to ET-1. Treatment of diseased PAs from PH patients with a CD47 blocking antibody improved sensitivity to vasodilators. Hypoxic wild type (WT) mice developed PH and displayed upregulation of pulmonary TSP1, CD47, and ET-1/ETA concurrent with down regulation of the transcription factor cell homolog of the v-myc oncogene (cMyc). In contrast, PH was attenuated in hypoxic CD47-/- mice while pulmonary TSP1 and ET-1/ETA were unchanged and cMyc was overexpressed. In CD47-/- pulmonary endothelial cells cMyc was increased and ET-1 decreased. In CD47+/+ cells, forced induction of cMyc suppressed ET-1 transcript, whereas suppression of cMyc increased ET-1 signaling. Furthermore, disrupting TSP1-CD47 signaling in pulmonary smooth muscle cells abrogated ET-1-stimulated hypertrophy. Finally, a CD47 antibody given 2 weeks after monocrotaline challenge in rats upregulated pulmonary cMyc and improved aberrations in PH-associated cardiopulmonary parameters.ConclusionsIn pre-clinical models of PH CD47 targets cMyc to increase ET-1 signaling. In clinical PH TSP1-CD47 is upregulated, and in both, contributes to pulmonary arterial vasculopathy and dysfunction.

| S-EPMC5220673 | biostudies-literature

Regulation of senescence escape by TSP1 and CD47 following chemotherapy treatment.

Project description:Senescence is a tumor-suppressive mechanism induced by telomere shortening, oncogenes, or chemotherapy treatment. Although it is clear that this suppressive pathway leads to a permanent arrest in primary cells, this might not be the case in cancer cells that have inactivated their suppressive pathways. We have recently shown that subpopulations of cells can escape chemotherapy-mediated senescence and emerge as more transformed cells that induce tumor formation, resist anoikis, and are more invasive. In this study, we characterized this emergence and showed that senescent cells favor tumor growth and metastasis, in vitro and in vivo. Senescence escape was regulated by secreted proteins produced during emergence. Among these, we identified thrombospondin-1 (TSP1), a protein produced by senescent cells that prevented senescence escape. Using SWATH quantitative proteomic analysis, we found that TSP1 can be detected in the serum of patients suffering from triple-negative breast cancer and that its low expression was associated with treatment failure. The results also indicate that senescence escape is explained by the emergence of CD47low cells that express a reduced level of CD47, the TSP1 receptor. The results show that CD47 expression is regulated by p21waf1. The cell cycle inhibitor was sufficient to maintain senescence since its downregulation in senescent cells increased cell emergence. This leads to the upregulation of Myc, which then binds to the CD47 promoter to repress its expression, allowing the generation of CD47low cells that escape the suppressive arrest. Altogether, these results uncovered a new function for TSP1 and CD47 in the control of chemotherapy-mediated senescence.

| S-EPMC6393582 | biostudies-literature

The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

Project description:Senescent cells withdraw from the cell cycle and do not proliferate. The prevalence of senescent compared to normally functioning parenchymal cells increases with age, impairing tissue and organ homeostasis. A contentious principle governing this process has been the redox theory of aging. We linked matricellular protein thrombospondin 1 (TSP1) and its receptor CD47 to the activation of NADPH oxidase 1 (Nox1), but not of the other closely related Nox isoforms, and associated oxidative stress, and to senescence in human cells and aged tissue. In human endothelial cells, TSP1 promoted senescence and attenuated cell cycle progression and proliferation. At the molecular level, TSP1 increased Nox1-dependent generation of reactive oxygen species (ROS), leading to the increased abundance of the transcription factor p53. p53 mediated a DNA damage response that led to senescence through Rb and p21cip, both of which inhibit cell cycle progression. Nox1 inhibition blocked the ability of TSP1 to increase p53 nuclear localization and p21cip abundance and its ability to promote senescence. Mice lacking TSP1 showed decreases in ROS production, p21cip expression, p53 activity, and aging-induced senescence. Conversely, lung tissue from aging humans displayed increases in the abundance of vascular TSP1, Nox1, p53, and p21cip Finally, genetic ablation or pharmacological blockade of Nox1 in human endothelial cells attenuated TSP1-mediated ROS generation, restored cell cycle progression, and protected against senescence. Together, our results provide insights into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence and suggest potential targets for controlling the aging process at the molecular level.

| S-EPMC5679204 | biostudies-literature

Recombinant TSP1 cleaved by HTRA1 (LCMS)

Project description:HTRA1 proteolysis of TSP1 inhibits immune suppression in age-related macular degeneration. A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age related macular degeneration (AMD), a major cause of blindness affecting millions of individuals worldwide. Here we show that monocytes (Mo) from homozygous carriers of the 10q26 AMD-risk haplotype overexpress the High-Temperature Requirement A Serine Peptidase 1 (HTRA1), which locates to mononuclear phagocytes (MP) in eyes of patients with AMD. Elevated HTRA1 led to significant subretinal Mo persistence and promoted pathogenic MP accumulation due to the hydrolysis of Thrombospondin 1 (TSP1). Mechanistically, we demonstrate that HTRA1 separates TSP1’s two CD47- binding Valine-Valine-Methionine-sites that are necessary for efficient CD47 activation and repression of osteopontin (OPN) secretion, a mediator of inflammation and wound healing. Accordingly, OPN was overexpressed in early Mo-derived macrophages in 10q26 risk carriers and OPN deletion or inhibition fully reversed HTRA1- induced pathogenic MP persistence. Our observations provide new insights into the molecular mechanisms of MP accumulation in inflammation and show that HTRA1 resistant CD47 agonists and OPN inhibitors can provide a powerful tool to reverse HTRA1’s pro-inflammatory effect and restore retinal immune suppression in AMD, critical for retinal homeostasis.

2020-07-25 | PXD020546 | Pride

Modulation of the pharmacokinetics of soluble ACE2 decoy receptors through glycosylation.

Project description:The Spike of SARS-CoV-2 recognizes a transmembrane protease, angiotensin-converting enzyme 2 (ACE2), on host cells to initiate infection. Soluble derivatives of ACE2, in which Spike affinity is enhanced and the protein is fused to Fc of an immunoglobulin, are potent decoy receptors that reduce disease in animal models of COVID-19. Mutations were introduced into an ACE2 decoy receptor, including adding custom N-glycosylation sites and a cavity-filling substitution together with Fc modifications, which increased the decoy's catalytic activity and provided small to moderate enhancements of pharmacokinetics following intravenous and subcutaneous administration in humanized FcRn mice. Most prominently, sialylation of native glycans increases exposures by orders of magnitude, and the optimized decoy is therapeutically efficacious in a mouse COVID-19 model. Ultimately, an engineered and highly sialylated decoy receptor produced using methods suitable for manufacture of representative drug substance has high exposure with a 5- to 9-day half-life. Finally, peptide epitopes at mutated sites in the decoys generally have low binding to common HLA class II alleles and the predicted immunogenicity risk is low. Overall, glycosylation is a critical molecular attribute of ACE2 decoy receptors and modifications that combine tighter blocking of Spike with enhanced pharmacokinetics elevate this class of molecules as viable drug candidates.

| S-EPMC11342882 | biostudies-literature

Inhibitory receptor CD47 binding to plasma TSP1 suppresses NK-cell IFN-γ production via activating the JAK/STAT3 pathway during HIV infection.

Project description:BackgroundNatural killer (NK) cells play an important first-line role against tumour and viral infections and are regulated by inhibitory receptor expression. Among these inhibitory receptors, the expression, function, and mechanism of cluster of differentiation 47 (CD47) on NK cells during human immunodeficiency virus (HIV) infection remain unclear.MethodsFresh peripheral blood mononuclear cells (PBMCs) were collected from people living with HIV (PLWH) and HIV negative controls (NC) subjects. Soluble ligand expression levels of CD47 were measured using ELISA. HIV viral proteins or Toll-like receptor 7/8 (TLR7/8) agonist was used to investigate the mechanisms underlying the upregulation of CD47 expression. The effect of CD47 on NK cell activation, proliferation, and function were evaluated by flow cytometry. RNA-seq was used to identify downstream pathways for CD47 and its ligand interactions. A small molecule inhibitor was used to restore the inhibition of NK cell function by CD47 signalling.ResultsCD47 expression was highly upregulated on the NK cells from PLWH, which could be due to activation of the Toll-like receptor 7/8 (TLR7/8) pathway. Compared with NC subjects, PLWH subjects exhibited elevated levels of CD47 ligands, thrombospondin-1 (TSP1), and counter ligand signal regulatory protein-α (SIRPα). The TSP1-CD47 axis drives the suppression of interferon gamma (IFN-γ) production and the activation of the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway in NK cells. After treatment with a STAT3 inhibitor, the NK cells from PLWH showed significantly improved IFN-γ production.ConclusionsThe current data indicate that the binding of the inhibitory receptor CD47 to plasma TSP1 suppresses NK cell IFN-γ production by activating the JAK/STAT3 pathway during HIV infection. Our results suggest that CD47 and its related signalling pathways could be targets for improving NK cell function in people living with HIV.

| S-EPMC10688093 | biostudies-literature

Pharmacokinetics of recombinant human soluble thrombomodulin in disseminated intravascular coagulation patients with acute renal dysfunction.

Project description:Recombinant human soluble thrombomodulin (ART-123) is a novel anticoagulant for patients with disseminated intravascular coagulation (DIC). It is widely used in clinical settings throughout Japan. Furthermore, a global Phase 3 study is currently being conducted. In healthy subjects, ART-123 is excreted mainly via the kidneys. Therefore, ART-123 dose decrease was recommended in DIC patients with severe renal dysfunction. However, the pharmacokinetics of ART-123 in DIC patients with severe acute renal dysfunction has not been elucidated. In an open-label, multicentre, prospective, clinical pharmacological study, we investigated the pharmacokinetics and safety of ART-123 upon repeated administration to DIC patients. ART-123 was administered to patients at a dose of 130 or 380 U/kg/day for six consecutive days. Plasma concentrations of ART-123 were measured at 21 time points until eight days after the final administration. Urinary excretion rates during the first 24 hours (h) were calculated. Patient renal functions were evaluated by measuring 24-h creatinine clearance (Ccr). Forty-three patients were enrolled in the present study. The urinary excretion rates of ART-123 correlated closely with 24-h Ccr. Total body clearance of ART-123 was also weakly related with 24-h Ccr. However, the plasma concentrations of ART-123 were not considerably different among patients with different renal function. Two patients had subcutaneous haemorrhage as an adverse event related to ART-123. In conclusion, plasma concentrations of ART-123 may not be different among patients with different renal functions. ART-123 was well tolerated in these patients.

| S-EPMC5442600 | biostudies-other

A novel decoy strategy for polymyxin resistance in Acinetobacter baumannii.

Project description:Modification of the outer membrane charge by a polymyxin B (PMB)-induced PmrAB two-component system appears to be a dominant phenomenon in PMB-resistant Acinetobacter baumannii. PMB-resistant variants and many clinical isolates also appeared to produce outer membrane vesicles (OMVs). Genomic, transcriptomic, and proteomic analyses revealed that upregulation of the pmr operon and decreased membrane-linkage proteins (OmpA, OmpW, and BamE) are linked to overproduction of OMVs, which also promoted enhanced biofilm formation. The addition of OMVs from PMB-resistant variants into the cultures of PMB-susceptible A. baumannii and the clinical isolates protected these susceptible bacteria from PMB. Taxonomic profiling of in vitro human gut microbiomes under anaerobic conditions demonstrated that OMVs completely protected the microbial community against PMB treatment. A Galleria mellonella-infection model with PMB treatment showed that OMVs increased the mortality rate of larvae by protecting A. baumannii from PMB. Taken together, OMVs released from A. baumannii functioned as decoys against PMB.

| S-EPMC8324293 | biostudies-literature

Target-small decoy search strategy for false discovery rate estimation.

Project description:BackgroundOne of the most important steps in peptide identification is to estimate the false discovery rate (FDR). The most commonly used method for estimating FDR is the target-decoy search strategy (TDS). While this method is simple and effective, it is time/space-inefficient because it searches a database that is twice as large as the original protein database. This inefficiency problem becomes more evident as protein databases get bigger and bigger. We propose a target-small decoy search strategy and present a rigorous verification that it reduces the database size and search time while retaining the accuracy of target-decoy search strategy (TDS).ResultsWe show that peptide spectrum matches (PSMs) obtained at 1% FDR in TDS overlap ~ 99% with those in our method. (Considering that 1% FDR is used, 99% overlap means our method is very accurate.) Moreover, our method is more time/space-efficient than TDS. The search time of our method is reduced to only 1/4 of that of TDS when UniProt and its 1/8 decoy database are used.ConclusionsWe demonstrate that our method is almost as accurate as TDS and more time/space-efficient than TDS. Since the efficiency of our method is more evident as the database size increases, our method is expected to be useful for identifying peptides in proteogenomics databases constructed from inflated databases using genomic data.

| S-EPMC6708216 | biostudies-literature

Induction of antagonistic soluble decoy receptor tyrosine kinases by intronic polyA activation.

Project description:Alternative intronic polyadenylation (IPA) can generate truncated protein isoforms with significantly altered functions. Here, we describe 31 dominant-negative, secreted variant isoforms of receptor tyrosine kinases (RTKs) that are produced by activation of intronic poly(A) sites. We show that blocking U1-snRNP can activate IPA, indicating a larger role for U1-snRNP in RNA surveillance. Moreover, we report the development of an antisense-based method to effectively and specifically activate expression of individual soluble decoy RTKs (sdRTKs) to alter signaling, with potential therapeutic implications. In particular, a quantitative switch from signal transducing full-length vascular endothelial growth factor receptor-2 (VEGFR2/KDR) to a dominant-negative sKDR results in a strong antiangiogenic effect both on directly targeted cells and on naive cells exposed to conditioned media, suggesting a role for this approach in interfering with angiogenic paracrine and autocrine loops.

| S-EPMC3781938 | biostudies-literature

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