Thrombospondin Type Research Articles

A Genome-Wide Association Study for Susceptibility to Axial Length in Highly Myopic Eyes.

The online version contains supplementary material available at 10.1007/s43657-022-00082-x.

Recurrent Cryptogenic Stroke in a Young Woman: Congenital Thrombotic Thrombocytopenic Purpura Unmasked

Cryptogenic stroke of undetermined cause should warrant an exhaustive neurologic and cardiovascular workup. If no cause is identified, additional investigations should be individualized on the basis of clinical history and objective findings. Herein, we present the case of a young patient with a personal and family history of cryptogenic stroke who was investigated for thrombophilia. Transient thrombocytopenia and peripheral blood schistocytes led to an eventual diagnosis of congenital thrombotic thrombocytopenic purpura, which is characterized by vaso-occlusive end-organ complications. The diagnosis is confirmed by undetectable ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13) activity.

ADAMTS13 inhibition to treat acquired von Willebrand syndrome during mechanical circulatory support device implantation

BackgroundAcquired von Willebrand syndrome (aVWS) is common in patients with mechanical circulatory support (MCS) devices. In these patients, the high shear stress in the device leads to increased shear‐induced proteolysis of von Willebrand factor (VWF) by A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, number 13 (ADAMTS13). As a result, the high molecular weight (HMW) VWF multimers are lost, leading to a decreased VWF function and impaired hemostasis that could explain the bleeding complications that are frequently observed in these patients. To counteract this abnormal VWF degradation by ADAMTS13, we developed a novel targeted therapy, using an anti‐ADAMTS13 monoclonal antibody (mAb) that inhibits the shear‐induced proteolysis of VWF by ADAMTS13. MethodsHuman or bovine blood was circulated through in vitro MCS device systems with either inhibitory anti‐ADAMTS13 mAb 3H9 or 17C7 (20 μg/ml) or control anti‐ADAMTS13 mAb 5C11 or phosphate buffered saline (PBS). VWF multimers and function (collagen binding activity) were determined at different time points. Next, Impella pumps were implanted in calves and the calves were injected with PBS and subsequently treated with mAb 17C7. VWF, ADAMTS13, and blood parameters were determined. ResultsWe demonstrated that blocking ADAMTS13 could prevent the loss of HMW VWF multimers in in vitro MCS device systems. Importantly, our antibody could reverse aVWS in a preclinical Impella‐induced aVWS calf model. ConclusionHence, inhibition of ADAMTS13 could become a novel therapeutic strategy to manage aVWS in MCS device patients.

Prediagnostic Appearance of Thrombospondin Type-1 Domain 7A Autoantibodies in Membranous Nephropathy.

Key Points The entire extracellular domain of thrombospondin type-1 domain 7A (THSD7A) in the luciferase immunoprecipitation system immunoassay was required to detect autoantibodies with high sensitivity in membranous nephropathy (MN).In THSD7A-seropositive MN patients, changes in antibody levels precede changes in clinical status.Seropositive THSD7A antibodies were detected in some patients with MN considered to be secondary to autoimmunity or cancer. Background Pathogenic autoantibodies against thrombospondin type-1 domain 7A (THSD7A) are present in approximately 3% of patients with membranous nephropathy (MN). Compared with PLA2R antibodies, less is known about THSD7A autoantibodies (ABs) because of the relative rarity and the lack of a commercially available quantitative immunoassay. Methods In this study, we describe the development and validation of a highly quantitative luciferase immunoprecipitation system (LIPS) assay for detecting THSD7A ABs and used it to study dominant THSD7A epitopes, disease associations, and monitoring disease activity. The Department of Defense Serum Repository (DODSR) was then used to analyze THSD7A AB in 371 longitudinal serum samples collected before clinical diagnosis of MN from 110 PLA2R-negative MN subjects. Results LIPS analysis demonstrated that a near full-length THSD7A (amino acids 1–1656) detected robust autoantibody levels in all known seropositive MN patients with 100% sensitivity and specificity compared with ELISA and/or Western blotting. Most of the THSD7A-seropositive subjects in our pilot cohort had evidence of coexisting autoimmunity or cancer. Moreover, three THSD7A-seropositive patients undergoing immunosuppressive therapy showed longitudinal autoantibody levels that tracked clinical status. Additional epitope analysis of two smaller protein THSD7A fragments spanning amino acids 1-416 and 1-671 demonstrated lower sensitivity of 32% and 44%, respectively. In the DODSR cohort, THSD7A seropositivity was detected in 4.5% of PLA2R-negative MN patients. In one primary and in one secondary MN-associated with cancer, THSD7A ABs were detectable <1 month before biopsy-proven diagnosis. In addition, three patients with lupus membranous nephropathy had detectable THSD7A ABs years before hypoalbuminemia and biopsy-proven diagnosis. Conclusions Although further studies are needed to explore the significance of THSD7A ABs in lupus membranous nephropathy, this study describes a novel, highly sensitive LIPS immunoassay for detecting THSD7A ABs and adds to the existing literature on THSD7A-associated MN. Clinical Trial registry name and registration number: NCT00977977; registration date: September 16, 2009.

Levels and Activity of Von Willebrand Factor in Patients with Hereditary Thrombotic Thrombocytopenic Purpura

Background: Mutations in the ADAMTS-13 gene in patients with hereditary thrombotic thrombocytopenic purpura interfere with production or function of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) enzyme and increase levels of highly active large von Willebrand factor (vWF) multimers. Recent data from a cohort of hTTP patients carrying cDNA.4477_4477delA mutation in ADAMTS-13 gene suggested a discrepancy between ADAMTS13 activity and severity of clinical presentation. We proposed that the levels and activity of vWF might be elevated and associated with disease severity. In the present study we evaluated the levels and activity of vWF from homozygous cDNA.4477_4477delA mutation carriers in comparison to an historical cohort. Methods: In this retrospective cohort study all patients were diagnosed and treated at Soroka University Medical Center, Beer Sheba, Israel. Diagnosis of hTTP was confirmed by abnormal low levels of ADAMTS13, lack of anti-ADAMTS13 antibodies and positive testing for cDNA.4477_4477delA mutation. Patients' data were collected prospectively, while pre-enrollment data were collected retrospectively from the hospital records. The tests for von Willebrand factor activity (vWF:RCof) and von Willebrand factor antigen (vWF:Ag) were performed on non-pregnant patients and before FFP transfusions were initiated. Patients' vWF:RCof and vWF:Ag levels were compared to the values from previously published data of healthy subjects (Kilercik et al, Biological variations of ADAMTS13 and von Willebrand factor in human adults, Biochem. Medica. 24 [2014] 138-145). The data were analyzed using descriptive variables. vWF:RCof and vWF:Ag levels were compared using Mann-Whitney test. Results: Study population included 18 adults (15 female and three male patients) from two related families, all homozygous for c.3772 delA mutation. The age at initial presentation ranged from 18 to 48 years (mean 28.7±8.8). 17 patients were identified through episodes of microangiopathic hemolytic anemia (MAHA), thrombocytopenia, vascular and pregnancy complications (see Figure 1B). One subject was detected due to family history as part of family screening. Symptomatic patients received FFP transfusions every two weeks. Pregnant patients received FFP transfusions at a dose of two units every two weeks until the week 20 and thereafter two units weekly until delivery. All patients responded well to FFP transfusions. We did not observe hTTP-related mortality among study participants. Figure 1A shows the comparison with an historic control group. The control group had similar size (19 vs. 18 subjects in the study group). The patients in our study group were slightly older and had a female predominance (83.3%), while the control group had 47% females and 53% males. All 18 patients in our cohort had blood group A, 15 (83%) have been Rh positive. 14 patients (82%, 12 females and two male) were thrombocytopenic on initial presentation (median, 28 x103/µL, interquartile range [IQR] 14 x103/µL - 144 x103/µL). 13 patients (73%) were anemic (median hemoglobin 10.2 gr/dL, IQR 7.6 gr/dL - 10.9 gr/dL). Lactate dehydrogenase (LDH) (median, 1425 U/L, IQR 915 U/L - 1912 U/L) was elevated in 16 patients (89%) during initial presentation. Seven patients (39%) had a schistocyte count ≥1% on peripheral blood smear. Values of vWF antigen (vWF:Ag) (median 209.3% [range 141.5-240.5%] vs. 104% [range 86-116%], p value <0.001) and vWF activity (vWF:RCof) (median 130.6% [range 102.6-139%] vs. 83% [range 77-99%], P value <0.001) were significantly higher in the study group than in healthy controls, respectively (Figure 1A, 1C). Conclusions: Our results demonstrate that the levels of vWF antigen and activity in patients with hTTP are significantly higher than in healthy controls. These findings prompt further evaluation of vWF as a possible biomarker for the severity of hTTP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Clinical Significance of the Glomerular Biomarker Soluble Urokinase-Type Plasminogen Activator Receptor (suPAR) in Renal Function of Caucasian Patients with Compound Heterozygosity for Hb S and β-Thalassemia

Background: A wide spectrum of glomerular lesions has been described in patients with Sickle Cell Disease (SCD). The most frequently identified morphologic lesion associated with SCD is perihilar Focal Segmental Glomerulosclerosis (FSGS). Glomerular ischemia leads to a compensatory increase in renal blood flow and glomerular filtration rate (GFR); such hyperfiltration, combined with glomerular hypertrophy, probably contributes to glomerulosclerosis. As glomerulosclerosis becomes more extensive, GFR starts to decrease. The glomerular biomarker suPAR is the circulating form of a glycosyl-phosphatidylinositol-anchored three domain membrane protein that is expressed on a variety of cells, including immunologically active cells, endothelial cells, and podocytes. suPAR has been implicated in the pathogenesis of kidney disease, specifically FSGS, through interference with podocyte migration and apoptosis. Recently, suPAR was associated also with subsequent acute kidney injury. We aimed to investigate a possible link between suPAR serum levels and renal function decline for the first time in Caucasian patients with compound heterozygosity for HbS and β-thalassemia (HbS/βthal). Patients and Methods: Ιn the study were included 138 Caucasian adult patients with HbS/βthal at steady phase, and 20 healthy individuals of similar age and gender who served as controls. suPAR concentration was measured with an immunoenzymatic assay (ViroGates, Denmark) along with a series of other blood chemistry markers of erythropoiesis, renal, endothelial and cardiac function, such as: Neutrophil Gelatinase Associated Lipocalin (NGAL), Interleukin-18 (IL-18), Retinol Binding Protein-4 (RBP-4), Fatty Acid Binding Protein-4 (FABP-4), von Willebrand factor antigen (VWF:antigen), D-Dimers, Α Disintegrin and Metalloproteinase with Thrombospondin Type 1 Motif 13 (ADAMTS-13), hs-Troponin-T (hs-TnT), Placental Growth Factor (PlGF) and Growth Differentiation Factor-15 (GDF-15), measured with immunoenzymatic and electrochemiluminescence immunoassays. eGFR values were calculated using the CKD-EPI (Cystatin C and/or Creatinine equations. Results: We found that: a) suPAR levels were higher in patients with HbS/βthal compared to controls, 4.2±2.7pg/mL (1.2-18.2pg/mL) vs 1.9±0.4pg/mL (1.0-2.4pg/mL), p<0.001; b) No correlation was found with hydroxycarbamide treatment (p>0.1), while a weak correlation was found between suPAR levels and HbS/βthal genotype (r=-0.256, p=0.012). c) suPAR levels correlated significantly with eGFR values r=-0.657,p<0.001 and CKD stage (ANOVA p<0.001); 70% of the patients with hyperfiltration and stage-1 CKD had increased suPAR levels; d) suPAR levels correlated significantly with patients’ values of age r=0.477, p<0.001, Hb r= -0.493, p<0.001, Reticulocyte production Index r=0.367, p=0.002, ferritin r=0.459, p<0.001 and uric acid r=0.215, p<0.01, while no correlation was found with LDH r=0.149, p>0.1. e) Comparing the diagnostic significance of suPAR with other parameters of tubular-glomerular activation such as NGAL, IL-18, RBP-4, FABP-4, GDF-15, only the tubular marker NGAL has slightly superior significance, while none of these renal markers was increased in patients with hyperfiltration. f) Nevertheless, suPAR levels may reflect defects to more systems as correlated significance with markers of endothelial dysfunction-hemostatic system: vWF:antigen r=0.466, p<0.001, D-dimer r=0.344, p<0.001), ADAMTS-13/VWF ratio r=-0.485, p<0.001, cardiac injury and stress: hs-troponin-T r=0.519, p<0.001 and GDF-15 r=0.453, p<0.001) and angiogenesis: PlGF r=0.429, p<0.001 was found. Conclusions: To our knowledge this is the first report of the evaluation of a glomelurar biomarker such as suPAR in Caucasian patients with HbS/βthal, showing higher levels in these patients due to possible destruction of podocytes. As more than 70% of patients with hyperfiltration and stage-1 CKD, had increased suPAR levels, we suggest that suPAR could be a prognostic marker of renal injuries in patients with SCD particular in those patients with hyperfiltration and stage-1 CKD. However, this biomarker should be more investigated in order to determine its clinical significance.

Rapid Diagnosis of Acute Thrombotic Thrombocytopenic Purpura (TTP) Using a New Fully Automated Chemiluminescent Assay for Adamts-13 Activity. Clinical Performance and Cost-Effectiveness Analysis

Background: Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy (TMA) that can be caused by congenital or acquired severe deficiency of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13), an enzyme that cleaves von Willebrand factor (VWF). ADAMTS-13 deficiency leads to the accumulation of ultra-large VWF multimers, leading to platelet aggregation and microthrombi formation. Aside clinical symptoms, the diagnosis of TTP is based on laboratory parameters such as thrombocytopenia, hemolytic anemia with the presence of schistocytes, and severely reduced activity of ADAMTS-13 (<10%). As TTP is a life-threatening condition characterized by microvascular thrombosis, it is of great importance to shorten the time to the treatment, and according to the French recommendations, patients with suspected TTP must be treated with the triplet therapy including plasma exchange (PEX), rituximab, and caplacizumab, plus corticosteroids until the results of ADAMTS-13 was obtained. As this therapy must be pursued only in the case of deficiency, a rapid ADAMTS-13 testing is critical for early diagnosis and optimal management of acute TTP. Design and aims of the study: We first retrospectively compared the performance of a rapid fully automated chemiluminescent assay (HemosIL AcuStar ADAMTS-13) to those of the FRETS-VWF73 assay in the diagnosis of TTP. Then, we compared the cost-effectiveness of a strategy based on the on-site measurement of ADAMTS-13 activity using the AcuStar assay vs. its centralized measurement in a reference laboratory, as it is currently the case at our institution. For that purpose, we created two scenarios based on either the local measurement of ADAMTS-13 activity with a maximum time-to-result of 16 hours (assay performed during daytime, 7/7) or its centralized measurement with a median time-to-result of 4 days (range: 2-8), as demonstrated from the 2018-2021 estimates. The triplet therapy was hypothesized to be started on admission and stopped if ADAMTS-13 activity was >10%. In case of ADAMTS-13 activity <10%, an inhibitor screening would be performed at the reference laboratory, as this assay was not expected to be locally implemented. For the calculation, we considered the costs of reagent, shipping/handling of the samples to the reference laboratory, laboratory staff workload, reimbursement rates, and treatments administered in patients with suspected TTP until availability of the ADAMTS-13 result. Results: In the plasma from 56 patients with acute TMA, ADAMTS-13 activity was not significantly different when evaluated using the HemosIL AcuStar ADAMTS-13 and the FRETS-VWF73 assays [median=72.6% (range: 0.2-126) vs. 79% (range: 5-150); p=0.08]. The concordance of test results as whether ADAMTS-13 activity was below (n=5) or above the threshold of 10% using the 2 assays was excellent (kappa=1.00), allowing an accurate diagnosis of the 5 patients with confirmed TTP. Based on the local activity between 2018 and 2021, there was a mean of 60 prescriptions of ADAMTS-13 activity per year. Most were for the follow-up of TTP patients and for patients with non-TTP TMA, whereas 7 were for the diagnosis of acute TTP, as suggested by the French score, which was confirmed in 4 cases (mean values). As shown in the Table, the local measurement of ADAMTS-13 activity was found to be cost-effective with a 16%-reduction of the costs compared to its centralized measurement in the 7 patients with suspected TTP during the first 4 days. This equates to 4,508 € saved per patient with suspected acute TTP. Conclusions: Together with its short turnaround time (33 min) and its full automation, our results suggest that the HemosIL AcuStar ADAMTS-13 activity assay could be an assay of choice to rapidly measure ADAMTS-13 activity in the plasma and thus to establish the diagnosis of acute TTP in emergency settings. Considering the local costs, using the strategy based on the local evaluation of ADAMTS-13 activity led to a 16%-reduction of the overall costs during the first 4 days after admission of the 7 patients with suspected acute TTP. Moreover, by dramatically decreasing the time-to-result (<1 day vs. 4 days), unnecessary potentially harmful and expensive treatments could be withheld earlier in patients in whom the diagnosis of TTP was ruled out based on an ADAMTS-13 activity >10%. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase 3 Study to Evaluate the Efficacy and Safety of Caplacizumab without First-Line Therapeutic Plasma Exchange in Adults with Immune-Mediated Thrombotic Thrombocytopenic Purpura

Background: Caplacizumab (CPLZ) is a von Willebrand factor-directed antibody fragment that is indicated, in combination with therapeutic plasma exchange (TPE) and immunosuppressive therapy (IST), for the treatment of immune-mediated TTP (iTTP), also known as acquired thrombotic thrombocytopenic purpura (aTTP). While TPE is considered a mainstay of iTTP treatment, it is burdensome and associated with complications. Although reports from real-world clinical practice suggest the efficacy of TPE-free CPLZ regimens in iTTP, the use of CPLZ without first-line TPE has not been evaluated in the clinical trial setting. Aim: To evaluate the efficacy and safety of CPLZ in combination with IST without first-line TPE in adults with iTTP. Methods: MAYARI (NCT05468320) is a Phase 3, single-arm, open-label, multicenter study (13 countries and 49 sites) in adults experiencing an acute episode of iTTP. Study Population: Adults with a clinical diagnosis of initial or recurrent iTTP, defined as thrombocytopenia (platelet count <100×109/L), microangiopathic hemolytic anemia, and a French thrombotic microangiopathy score of 1 or 2, are eligible for the study pending confirmation of diagnosis with an ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity level within 48 hours of enrollment. Exclusion criteria include other known etiologies of thrombocytopenia, serum creatinine >2.26 mg/dL if platelet count is >30×109/L, severe neurological or cardiac disease, clinically significant active bleeding or high bleeding risk, or concomitant use of chronic anticoagulant or antiplatelet therapy. Interventions: All enrolled participants will begin CPLZ and corticosteroid therapy immediately based on the clinical diagnosis of iTTP and a French score of 1 or 2. Anti-CD20 antibody therapy can be initiated after confirmation of iTTP diagnosis (ADAMTS13 <10%). If, after initiation of CPLZ therapy, baseline ADAMTS13 activity level is found to be 10%-20%, the investigator may use their judgment to decide whether to continue CPLZ. If ADAMTS13 level is found to be >20% CPLZ should be discontinued. Otherwise, CPLZ treatment will be continued until sustained ADAMTS13 activity level of ≥50% at 2 consecutive visits after platelet count normalization. The maximum duration allowed for treatment of the presenting episode is 12 weeks (on-treatment period). The post-treatment follow-up period will be 12 weeks. Participants will not receive TPE as first-line therapy, but TPE may be started after 24 hours for a lack of adequate response per prespecified criteria or clinical deterioration at any time point. Study Outcomes: The primary endpoint is the proportion of participants achieving remission without requiring TPE during the overall study period (Table). Secondary endpoints include proportion of participants achieving remission, proportion of participants requiring TPE, proportion of participants achieving a clinical response, time to initial platelet count response (≥150×109/L sustained for ≥2 days), proportion of participants with iTTP-related exacerbation or death, refractoriness, and overall mortality. Patient-reported clinical outcome assessments will also be utilized in this study to assess impact on quality of life measures. Safety and tolerability will also be assessed. Revised outcomes definitions from the International Working Group for iTTP will be utilized (Cuker et al. Blood. 2021;137[14]:1855-1861). Statistical Approach: An adequate number of participants will be enrolled to ensure at least 55 participants with ADAMTS13 activity levels <10% at baseline are available for analysis of the primary endpoint. It is anticipated that approximately 61 participants will be enrolled in the study. With the sample size of 55, assuming the true responder rate of participants who achieve remission without requiring TPE during the overall study period is 70%, the lower bound of 95% confidence interval would be 58%. Summary: The current standard of care in patients with iTTP includes a combination of TPE, IST, and CPLZ. This novel study will define the efficacy and safety of CPLZ and IST without first-line TPE in adults with iTTP. This regimen would avert the risks for substantial complications associated with TPE and represents a paradigm shift in the frontline management of iTTP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Preemptive cyclosporin A in immune-mediated thrombotic thrombocytopenic purpura.

Survivors of immune-mediated thrombotic thrombocytopenic purpura (iTTP) are exposed to clinical relapses when a disintegrin and metalloproteinase with thrombospondin type 1 repeats, member 13 (ADAMTS13) activity decreases during follow-up. Although preemptive rituximab usually improves ADAMTS13 activity in this context, 15% of patients experience refractoriness or intolerance to rituximab and require alternative strategies. Here, we addressed whether cyclosporine A (CSA) could improve ADAMTS13 activity and prevent clinical relapses in this context. We treated preemptively with CSA 14 iTTP patients who were unresponsive (n= 11) or intolerant (n= 3) to rituximab. All patients had a severe ADAMTS13 deficiency (activity <20%) and otherwise in clinical remission. ADAMTS13 activity normalized in almost all patients (n= 13, 93%), after a median time of 2.5months [IQR 1-6] following initiation. Median duration of CSA treatment was 17.5months [IQR 10-34]. ADAMTS13 activity further declined to undetectable values during follow-up in five patients, but retreatment with rituximab or CSA allowed a recovery in ADAMTS13 activity in three cases. CSA could be stopped durably in two patients, while two others experienced an ADAMTS13 relapse. Severe but reversible side effects requiring cessation of the treatment occurred in two patients. CSA provides high and sustained response rates in patients who are refractory or intolerant to rituximab, with acceptable adverse events.

ANGI-08. EPIGENETIC REACTIVATION OF BAI1 SUPPRESSES TUMOR INVASION, RECURRENCE, AND SPINAL METASTASIS BY PREVENTING TGFΒ1-INDUCED MESENCHYMAL SWITCH IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common and lethal type of malignant brain tumor in adults. GBM cells spread extensively in the brain and disseminate into the cerebrospinal fluid space, strongly restricting multimodal therapies. Acquiring a better knowledge of molecular defects underlying GBM invasion is essential for developing effective treatments.Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is a transmembrane receptor of the adhesion GPCR family widely expressed in the normal brain, but its expression is lost in the majority of GBM through epigenetic silencing and restoration of its expression can inhibit glioma growth. However, whether BAI1 loss is important for tumor invasion and the mesenchymal phenotype in GBM has not been investigated.Microarray analysis of the GBM TCGA dataset (restricted to IDHwt GBM; WHO 2021) revealed that low BAI1 mRNA expression correlates with elevated expression of many mesenchymal genes. Restoration of BAI1 expression in human GBM cells suppresses mesenchymal gene expression in culture and dramatically decreases brain tumor invasion in mice xenografts. Mechanistically, we found that the N-terminal thrombospondin type 1 repeat (TSR#1) of BAI1 inhibits the maturation process of TGFβ1, a key growth factor involved in EMT. BAI1 is silenced epigenetically in GBM cells by methylated CpG-binding protein MBD2, and its expression can be reactivated by KCC-07, a blood-brain barrier permeable MBD2 inhibitor. We found that restoration of BAI1 expression by KCC-07 treatment dramatically suppresses cell invasion in the brain and reduces leptomeningeal dissemination of GBM cells to the spine in mouse xenografts.These experiments demonstrate that epigenetic silencing of BAI1 is important for activating the GBM invasive phenotype through TGFβ1 pathway activation. This new tumor-suppressive pathway can be epigenetically targeted to reactivate BAI1 expression in GBM patients.

From the Discovery of ADAMTS13 to Current Understanding of Its Role in Health and Disease.

ADAMTS13 (a disintegrin-like metalloprotease domain with thrombospondin type 1 motif, member 13) is a protease of crucial importance in the regulation of the size of von Willebrand factor multimers. Very low ADAMTS13 activity levels result in thrombotic thrombocytopenic purpura, a rare and life-threatening disease. The mechanisms involved can either be acquired (immune-mediated thrombotic thrombocytopenic purpura [iTTP]) or congenital (cTTP, Upshaw-Schulman syndrome) caused by the autosomal recessive inheritance of disease-causing variants (DCVs) located along the ADAMTS13 gene, which is located in chromosome 9q34. Apart from its role in TTP, and as a regulator of microthrombosis, ADAMTS13 has begun to be identified as a prognostic and/or diagnostic marker of other diseases, such as those related to inflammatory processes, liver damage, metastasis of malignancies, sepsis, and different disorders related to angiogenesis. Since its first description almost 100 years ago, the improvement of laboratory tests and the description of novel DCVs along the ADAMTS13 gene have contributed to a better and faster diagnosis of patients under critical conditions. The ability of ADAMTS13 to dissolve platelet aggregates in vitro and its antithrombotic properties makes recombinant human ADAMTS13 treatment a potential therapeutic approach targeting not only patients with cTTP but also other medical conditions.

Identification of polyunsaturated fatty acids related key modules and genes in metabolic dysfunction-associated fatty liver disease using WGCNA analysis.

Polyunsaturated fatty acids (PUFAs) play important roles in the aetiology and pathogenesis of metabolic dysfunction-associated fatty liver disease (MAFLD). However, the underlying molecular mechanisms are not understood. We analysed a public GEO dataset, GSE89632, to identify differentially expressed genes (DEGs) in MAFLD. Weighted gene coexpression network analysis (WGCNA) was used to reveal the core gene regulation network and to explore the PUFA-related hub genes in MAFLD. We experimentally verified these genes by quantitative reverse transcription PCR in high-fat diet (HFD)-fed mice. A total of 286 common DEGs (89 upregulated; 197 downregulated), mostly related to inflammatory and immune responses, were identified. Six modules were constructed using WGCNA, and 2 modules showed significant correlations with PUFAs. After combining these 2 modules with DEGs, the top 10 hub genes were identified. We further established a MAFLD mouse model with liver steatosis, as proved by HE and Oil Red O staining. Of the hub genes, ADAM metallopeptidase with thrombospondin type 1 motif 1 (adamts1) (p = 0.005) and transforming growth factor β3 (tgfβ3) (p < 0.001) showed significantly lower mRNA expression in MAFLD in vivo. adamts1 and tgfβ3 bridged PUFAs and MAFLD, which might be potential causative genes and therapeutic targets of MAFLD.

Long-term risk of relapse in immune-mediated thrombotic thrombocytopenic purpura and the role of anti-CD20 therapy

AbstractDisease relapse is recognized as a risk in immune-mediated thrombotic thrombocytopenic purpura (iTTP) after treatment of the acute presenting episode. Identification of patients at risk of relapse and its patterns are yet to be clearly established. We reviewed patients with iTTP having had >3 years of follow-up over 10 years in the United Kingdom to identify patient characteristics for relapse, assess relapse rates and patterns, and response to anti-CD20 therapy in those with a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) relapses (ADAMTS13 activity of <20% without thrombocytopenia). We identified 443 patients demonstrating relapse rates of 40% at 5-year follow-up. At 10-year follow-up, no difference in relapse was observed irrespective of whether rituximab was used at acute presentation (P = .39). Black Caribbean ethnicity increased the risk of disease relapse in the British population. There was a distinct population of patients (6%) that relapsed early with subsequent frequent relapses occurring on average within 2 years (average time to relapse in subgroup, 1.7 years). Overall, nearly 60% of relapses described were ADAMTS13 relapses, with subsequent treatment reducing the risk of progression to clinical relapses. We demonstrate that iTTP diagnosed in the latter part of the study period had lower rates of clinical relapses (22.6% vs 11.1%, P = .0004) with the advent of regular monitoring and preemptive rituximab. In ADAMTS13 relapses, 96% responded to anti-CD20 therapy, achieving ADAMTS13 activity of >20%. Anti-CD20 therapy was demonstrated to be an effective long-term treatment regardless of relapse pattern and there was no loss of this treatment response after subsequent treatment episodes.

ADAMTS12, a novel prognostic predictor, promotes cell proliferation, migration, and invasion in head and neck squamous cell carcinoma.

The prognostic significance and potential carcinogenic mechanism of ADAM metallopeptidase with thrombospondin type 1 motif 12 (ADAMTS12) in head and neck squamous cell carcinoma (HNSC) remain unclear. Immunohistochemistry was used to analyze the correlation between ADAMTS12 protein expression and clinicopathological factors in tumor samples from 195 patients with HNSC. Based on clinicopathological data of patients, Cox regression and Kaplan-Meier analysis were used to identify the prognostic significance of the ADAMTS12 expression. The carcinogenicity of the ADAMTS12 in HNSC cells was analyzed by CCK-8 assay, the wound-healing assay, and transwell assays after transfection of ADAMTS12 overexpression or knock-down vector. The expression of ADAMTS12 was up-regulated in HNSC compared with normal tissue, related to pathology grade and lymph node metastasis of patients with HNSC, which was an independent prognostic factor. ADAMTS12 overexpression facilitated cell viability, invasion, and migration of HNSC cells, while ADAMTS12 knock-down had inverse results. Moreover, enrichment analysis, ADAMTS12 overexpression assay, and ADAMTS12 knock-down assay confirmed that ADAMTS12 mediated the activation of P13K/Akt pathway in HNSC. Our studies indicated that ADAMTS12 was a novel prognostic biomarker and potentially therapeutic target in HNSC.

Identification of Host Molecules Involved in the Proliferation of Nucleopolyhedrovirus in Bombyx mori.

The Bombyx mori nucleopolyhedrovirus (BmNPV), a foodborne infectious virus, is the pathogen causing nuclear polyhedrosis and high lethality in the silkworm. In this study, we characterized the molecules involved in BmNPV-silkworm interaction by RNA sequencing of the fat body isolated from the virus-susceptible strain P50. Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation showed that the upregulated differentially expressed genes (DEGs) were mainly involved in translation, signal transduction, folding, sorting, and degradation, as well as transport and catabolism, while the downregulated DEGs were predominantly enriched in the metabolism of carbohydrates, amino acids, and lipids at 72 h post BmNPV infection. Knockout of the upregulated somatomedin-B and thrombospondin type-1 domain-containing protein, probable allantoicase, trifunctional purine biosynthetic protein adenosine-3, and Psl and pyoverdine operon regulator inhibited the proliferation of BmNPV, while knockout of the downregulated clip domain serine protease 3 and carboxylesterase clade H, member 1 promoted it. The molecules herein identified provide a foundation for developing strategies and designing drugs against BmNPV.

Membranous nephropathy: Clearer pathology and mechanisms identify potential strategies for treatment.

Primary membranous nephropathy (PMN) is one of the common causes of adult-onset nephrotic syndrome and is characterized by autoantibodies against podocyte antigens causing in situ immune complex deposition. Much of our understanding of the disease mechanisms underpinning this kidney-limited autoimmune disease originally came from studies of Heymann nephritis, a rat model of PMN, where autoantibodies against megalin produced a similar disease phenotype though megalin is not implicated in human disease. In PMN, the major target antigen was identified to be M-type phospholipase A2 receptor 1 (PLA2R) in 2009. Further utilization of mass spectrometry on immunoprecipitated glomerular extracts and laser micro dissected glomeruli has allowed the rapid discovery of other antigens (thrombospondin type-1 domain-containing protein 7A, neural epidermal growth factor-like 1 protein, semaphorin 3B, protocadherin 7, high temperature requirement A serine peptidase 1, netrin G1) targeted by autoantibodies in PMN. Despite these major advances in our understanding of the pathophysiology of PMN, treatments remain non-specific, often ineffective, or toxic. In this review, we summarize our current understanding of the immune mechanisms driving PMN from animal models and clinical studies, and the implications on the development of future targeted therapeutic strategies.

Effect of tranexamic acid on endothelial von Willebrand factor/ADAMTS-13 response to in vitro shock conditions.

Traumatic/hemorrhagic shock, sepsis and other inflammatory processes lead to endothelial activation and a loss of the endothelial glycocalyx. von Willebrand factor (vWF) is an acute phase reactant that is released from endothelial cells and megakaryocytes. Stimulated but not basal vWF leads to significant formation of ultralarge multimers (ultralarge vWF [ULvWF]) and risk for thrombotic complications. Ultralarge vWF is cleaved by a disintegrin and metalloproteinase with a thrombospondin type motif 13 (ADAMTS 13); alterations in ULvWF/ADAMTS 13 ratio may contribute to trauma-induced coagulopathy. Salutary effects of tranexamic acid (TXA) on trauma-induced coagulopathy have been described. These effects appear apart from antifibrinolytic actions of TXA and include protection of the endothelial glycocalyx. Ultralarge vWF is in part anchored to the glycocalyx layer of the endothelium. Tranexamic acid protected the endothelial glycocalyx layer from degradation using a microfluidic model of the microcirculation subjected to hypoxia-reoxygenation and catecholamine excess. We hypothesized that TXA administration following shock conditions would impact the vWF-ADAMTS-13 axis by protecting the glycocalyx from degradation. This was studied in a endothelial microfluidic flow study. Human umbilical vein endothelial cells were established under flow conditions and subjected to biomimetic shock. Tranexamic acid was added after 90 minutes of perfusion. von Willebrand factor antigen and ADAMTS-13 activity were measured. Western blot analysis was performed for vWF characterization from perfusion media. Shock conditions increased vWF antigen and decreased ADAMTS 13 activity. Tranexamic acid ameliorated shock induced cleavage in the ADAMTS 13-vWF axis with a reduction of the thrombogenic ULvWF. These results suggest another mechanism whereby administration of TXA early following traumatic/hemorrhagic shock mitigates microvascular perfusion abnormalities and subsequent organ failure. The resultant effects on platelet adhesion and aggregation require further study.

Optic neuropathy associated with TGFβ dysregulation in mice with a glaucoma-causative mutation of ADAMTS10

Glaucoma is a neurodegenerative disease that causes irreversible blindness due to loss of retinal ganglion cells (RGCs) and their axons. We previously identified a G661R mutation of ADAMTS10 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motif 10) as the disease-causing mutation in a beagle model of glaucoma. ADAMTS10 is a secreted matrix metalloproteinase that belongs to the ADAMTS family which is involved in extracellular matrix (ECM) turnover. Previous studies have shown that ADAMTS10 binds fibrillin microfibrils, promotes their formation, and influences their fibrillin isoform composition. Here, we established a mouse model carrying the G661R mutation of ADAMTS10 (ADAMTS10G661R/G661R) to investigate its ocular phenotypes related to glaucoma and to explore possible functions of ADAMTS10. We found that ADAMTS10 was expressed in the inner retina and along RGC axons in the optic nerve. However, ADAMTS10 was not colocalized with fibrillin microfibrils in these tissues, suggesting fibrillin-independent function for ADAMTS10. In electroretinogram experiments, we found that ADAMTS10G661R/G661R mice had reduced amplitude of retinal responses to dim light stimulus, indicating RGC dysfunction. The reduced RGC function coincided with RGC axon structural changes manifested as smaller optic nerves and fewer optic nerve axons, which may contribute to glaucoma. The reduced number of optic nerve axons found for ADAMTS10G661R/G661R mice occurred early, suggesting developmental deficits. Subsequent experiments found increased apoptosis in the retina of ADAMTS10G661R/G661R mice during postnatal development, which could result in fewer RGCs produced, accounting for fewer optic nerve axons in adulthood. Consistent with a protective effect of transforming growth factor β (TGFβ) signaling against apoptosis during retinal development as shown previously by others, we found increased apoptosis accompanied by decreased TGFβ signaling in the developing retina of ADAMTS10G661R/G661R mice, suggesting a novel role for ADAMTS10 in regulating TGFβ signaling which could involve direct interaction between ADAMTS10 and latent TGFβ.

Anti‐inflammatory protective effect of ADAMTS‐13 in murine arthritis models

BackgroundPatients with rheumatoid arthritis (RA) have frequent thrombotic events with endothelial dysfunction. Von Willebrand factor (VWF) has been shown to bind neutrophil extracellular traps (NETs) and NETs are part of RA etiology. ObjectivesThis study aims to elucidate whether this prothrombotic status exacerbates inflammation in arthritis. Here we focus on the involvement of A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif, member 13 (ADAMTS‐13), an enzyme cleaving VWF and its effect on NET deposition and RA development. MethodsWe evaluated the influence of the Adamts13 gene and recombinant human ADAMTS‐13 (rhADAMTS‐13) on arthritis in the mouse models of collagen‐induced arthritis (CIA). We also assessed VWF and NETs in synovial tissue. ResultsSeveral Adamts13−/− mice developed arthritis, while Adamts13+/+ siblings did not. Synovial tissue from Adamts13−/− showed accumulation of NETs. Treatment of DBA/1 J mice, an arthritis‐susceptible strain, with well‐tolerated doses of rhADAMT13 reduced arthritis incidence and alleviated the severity of arthritis. Mice treated with rhADAMT13 presented less serum interleukin 6 and less bone erosion determined by micro‐computed tomography. The effects on arthritis severity were observed both when administering rhADAMTS‐13 prophylactically and also when given after arthritis has developed. In both conditions, rhADAMTS‐13 reduced VWF and NET deposition on proliferated synovial tissue evaluated by immunoblotting. ConclusionsOur results demonstrate the inhibitory role of Adamts13 in murine arthritis and the effectiveness of rhADAMTS‐13 treatment. Additionally, this study suggests that deposition of VWF in the synovium and subsequent pathogenic NET retention promotes arthritis. Treatment with rhADAMTS‐13 provides a potential therapeutic approach targeting inflammation and pro‐thrombotic state in arthritis.

The Efficacy of Cyclophosphamide Combined with Prednisone in Membranous Nephropathy Patients with Different Cytochrome P450 2B6 Gene Polymorphisms and Analysis of Factors Influencing the Efficacy.

ObjectiveTo investigate the efficacy of cyclophosphamide combined with prednisone in membranous nephropathy (MN) patients with different cytochrome P450 (CYP) 2B6 gene polymorphisms and explore the factors influencing the efficacy.MethodsWe performed a prospective and interventional study including 153 outpatients diagnosed with membranous nephropathy from April 2020 to June 2020 in the Bayannur Hospital. Based on the results of CYP2B6 gene polymorphisms, patients were divided into CYP2B6*4 group (785A>G) with 93 cases and CYP2B6*5 group (1459C>T) with 60 cases, and all patients were treated with cyclophosphamide and prednisone. The efficacy of the two groups was compared, and the serum levels of antibody against thrombospondin type-1 domain-containing 7A (THSD7A-Ab), 8-hydroxy-2’-deoxyguanosine (8-OHdG) and antibody against the M-type phospholipase A2 receptor (PLA2R-Ab) determined by the enzyme-linked immunosorbent method were analyzed in the two groups before and after treatment.ResultsAfter the treatment with cyclophosphamide and prednisone, serum levels of THSD7A-Ab, 8-OHdG, and PLA2R-Ab were higher in the CYP2B6*4 group than those in the CYP2B6*5 group (All three P<0.001). The univariate Logistic regression analysis showed that CYP2B6*4 (OR = 1.727, 95% CI: 1.028–2.654. P=0.012), CYP2B6*5 (OR = 1.802, 95% CI: 1.179–2.752. P=0.031), THSD7A-Ab (OR = 1.832, 95% CI: 0.871–2.348. P=0.023), 8-OHdG (OR = 1.661, 95% CI: 1.123–2.231. P=0.009), PLA2R-Ab (OR = 1.649, 95% CI: 1.083–2.761. P=0.017) were independent influencing factors on the efficacy of MN. The multivariate Logistic regression analysis showed that CYP2B6*4 (OR = 2.009, 95% CI: 1.327–2.703. P<0.001), CYP2B6*5 (OR = 3.009, 95% CI: 1.467–5.231. P=0.005), THSD7A-Ab (OR = 1.396, 95% CI: 1.002–1.897. P=0.019), 8-OHdG (OR = 0.704, 95% CI: 0.591–0.742. P<0.001), PLA2R-Ab (OR = 2.761, 95% CI: 1.231–3.918. P=0.017) were independent factors influencing the efficacy of cyclophosphamide and prednisone on MN.ConclusionCyclophosphamide combined with prednisone was effective in treating MN with different CYP2B6 gene polymorphisms. CYP2B6*4, CYP2B6*5, and serum levels of THSD7A-Ab, 8-OHdG, and PLA2R-Ab were independent influencing factors on the outcome of MN.