Alpha-2-Macroglobulin Attenuates Posttraumatic Osteoarthritis Cartilage Damage by Inhibiting Inflammatory Pathways With Modified Intra-articular Drilling in a Yucatan Minipig Model.

BACKGROUND: Posttraumatic osteoarthritis (PTOA) arises secondarily to joint trauma and is driven by catabolic inflammatory pathways. Alpha-2-macroglobulin (α2M) is a naturally occurring proteinase inhibitor found in human serum and synovial fluid that binds proteases as well as proinflammatory cytokines involved in the pathogenesis of PTOA. PURPOSE: (1) To investigate the therapeutic potential of intra-articular α2M injections during the acute stages of PTOA by inhibiting inflammatory pathways driven by the cytokines expressed by the synovium in a large preclinical Yucatan minipig model and (2) to determine if 3 intra-articular α2M injections have greater chondroprotective effects compared with 1 intra-articular injection. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 48 Yucatan minipigs were randomized into 4 groups (n = 12 each): (1) modified intra-articular drilling (mIAD) and saline (mIAD + saline), (2) mIAD and 1 intra-articular α2M injection (mIAD +α2M-1), (3) mIAD and 3 α2M injections (mIAD +α2M-3), and (4) sham control. Surgical hindlimbs were harvested at 15 weeks after surgery. Cartilage degeneration, synovial changes, inflammatory gene expression, and matrix metalloproteinase levels were evaluated. Gait asymmetry was measured before and after surgery using a pressure-sensing walkway system. RESULTS: Macroscopic lesion areas and microscopic cartilage degeneration scores were lower in the mIAD +α2M-1 and mIAD +α2M-3 groups compared with the mIAD + saline group (P < .05) and similar to those in the sham group (P > .05). Synovial membrane scores of the mIAD +α2M-1 and mIAD +α2M-3 groups were lower than that of the mIAD + saline group (P < .05) and higher than that of the sham group (P < .05). Interleukin-1 beta, nuclear factor kappa B, and tumor necrosis factor alpha mRNA expression in the synovium and matrix metalloproteinase-1 levels in synovial fluid were significantly lower in the mIAD +α2M-1 and mIAD +α2M-3 groups compared with the mIAD + saline group (P < .05). No significant differences were observed between the mIAD +α2M-1 and mIAD +α2M-3 groups for all measured outcomes. There were early changes in gait (P < .05) between preoperative and postoperative time points for the mIAD + saline, mIAD +α2M-1, and mIAD +α2M-3 groups that normalized by 15 weeks. CONCLUSION: Animals receiving early α2M treatment exhibited less cartilage damage, milder synovitis, and lower inflammation compared with animals with no α2M treatment. These results exemplify the early anti-inflammatory effects of α2M and provide evidence that intra-articular α2M injections may slow the progression of PTOA. CLINICAL RELEVANCE: In patients presenting with an acute joint injury, an early intervention with α2M may have the potential to reduce cartilage degeneration from catabolic pathways and delay the development of PTOA.

Insight into the molecular interaction between the anticancer drug, enzalutamide and human alpha-2-macroglobulin: Biochemical and biophysical approach.

The drug pharmacokinetics is affected upon binding with proteins, thus making drug-protein interactions crucial. This study investigated the interaction between enzalutamide and human major antiproteinase alpha-2-macroglobulin (α2M) by using multi spectroscopic and calorimetric techniques. The spectroscopic techniques such as circular dichroism (CD), intrinsic fluorescence, and UV-visible absorption were used to determine the mechanism of enzalutamide-α2M interaction. Studies on the quenching of fluorescence at three different temperatures showed that the enzalutamide-α2M complex is formed through static quenching mechanism. The change in microenvironment around tyrosine residues in protein was detected through synchronised fluorescence. The secondary structure of α2M was slightly altered by enzalutamide according to far UV-CD spectral analysis. Changes in position of amide I band in FTIR spectra further confirm the secondary structural alteration in α2M. According to thermodynamic characteristics such as fluorescence quenching and isothermal titration calorimetry (ITC), hydrogen bonds and hydrophobic interactions were involved in the interaction machanism. The ITC reiterated the exothermic and spontaneous nature of the interaction. The lower proteinase inhibitory activity of the α2M-enzalutamide conjugate as reflects the disruption of the native α2M structure upon interaction with enzalutamide.

Spontaneous Partial Regression of Fetal Lung Interstitial Tumor With A2M::ALK Rearrangement in a Neonate.

The differential diagnosis for neonatal primary lung masses includes developmental anomalies and congenital lung tumors. Fetal lung interstitial tumor (FLIT) is a rare benign mesenchymal lesion which presents either antenatally or within the first 3 months of age. FLIT is a circumscribed solid-cystic mass which histologically resembles the fetal lung during the canalicular stage at 20-24 weeks of gestation. It is composed of immature mesenchymal cells expanding the interstitium and irregular airspace-like structures. Of all published cases, only 1 identified an α2-macroglobulin (A2M)::anaplastic lymphoma kinase (ALK) fusion and all cases underwent surgical resection in the neonatal or infancy period. We present the second case of FLIT with an A2M::ALK fusion diagnosed postnatally in a neonate which partially regressed spontaneously during conservative management with interim resection at 39 months of age, and provide a review of the literature.

Alpha-2-Macroglobulin Is a Novel Anticancer Agent.

INTRODUCTION: Melanoma is the most aggressive skin cancer, with an increasing occurrence. Despite the recent important improvements due to novel immunotherapy approaches, when late diagnosed, melanoma prognosis is poor due to the metastatic progression and drug-resistance onset. Therefore, there is an urgent need to identify additional therapeutic targets. Melanoma invasive behavior is related to the activity of metalloproteases, able to degrade extracellular matrix leading to tumor dissemination. A recent study suggested that the most potent proteases inhibitor alpha-2-macroglobulin (A2MG) from plasma of hibernating fishes exerts potent antiproliferative effects. Our previous studies showed a significant reduction of A2MG in sera from mice/human melanoma models. METHODS: Gene and protein expression studies have been performed by using platforms and databases available online containing expression data from thousands of patients and healthy controls. RESULTS: We carried out an extensive bioinformatics analysis to evaluate the A2MG gene/protein expression on a large cohort of patients affected by many different cancer types, compared to healthy control subjects, and we found a highly significant difference of A2MG expression in 20 out of 31 cancer types (including melanoma) compared to healthy controls. Similar results were also confirmed at the proteomic level using another platform available online. Further, we found that higher A2MG expression is significantly related to overall survival in different cancers including melanoma. CONCLUSION: Our results strongly suggest A2MG as a novel molecular target in melanoma therapy, as well as in other cancer types.

Effect of Short PSG Peptides on Inflammatory Markers in Allogeneic Bone Marrow Cell Transplantation in Wistar Rats.

Short linear peptide fragments of placental trophoblastic ß1-glycoprotein (PSG) (YECE, YQCE, YVCS, and YACS) were studied in the context of their immunomodulatory effects at the level of inflammatory markers. The original host-versus-graft model was used in male Wistar rats without prior conditioning of recipient bone marrow. A composition of PSG peptide fragments was injected to animals after allogeneic transplantation of bone marrow cells in a dynamic experiment, inflammatory markers α1-acid glycoprotein (AGP, orosomucoid), α2-macroglobulin (α2M) were assayed by ELISA, and biochemical parameters (total protein, glucose, creatinine, and urea) were measured. The levels of α2M and AGP increased in response to allotransplantation, whereas administration of PSG peptides normalized serum α2M levels by the end of the experiment. The decrease in α2M level coincided with the independent effect of PSG peptide administration. The levels of total protein, glucose, creatinine, and urea in rat serum after allotransplantation were reduced throughout the experiment. Administration of PSG peptides contributed to normalization of serum total protein, creatinine, and urea levels by the end of the experiment. Administration of PSG peptides after allogeneic transplantation of bone marrow suspension contributed to normalization of the levels of α2M, total protein, creatinine, and urea, which can be interpreted as an anti-inflammatory effect of these peptides.

Pre-analytical handling conditions and protein marker recovery from urine extracellular vesicles for bladder cancer diagnosis.

Extracellular vesicles (EVs) contain a variety of biomolecules and provide information about the cells that produce them. EVs from cancer cells found in urine can be used as biomarkers to detect cancer, enabling early diagnosis and treatment. The potential of alpha-2-macroglobulin (A2M) and clusterin (CLU) as novel diagnostic urinary EV (uEV) biomarkers for bladder cancer (BC) was demonstrated previously. To validate the diagnostic value of these proteins in uEVs in a large BC cohort, urine handling conditions before uEV isolation should be optimized during sample transportation from medical centers. In this study, we analyzed the uEV protein quantity, EV particle number, and uEV-A2M/CLU after urine storage at 20°C and 4°C for 0-6 days, each. A2M and CLU levels in uEVs were relatively stable when stored at 4°C for a maximum of three days and at 20°C for up to 24 h, with minimal impact on analysis results. Interestingly, pre-processing to remove debris and cells by centrifugation and filtration of urine did not show any beneficial effects on the preservation of protein biomarkers of uEVs during storage. Here, the importance of optimizing shipping conditions to minimize the impact of pre-analytical handling on the uEVs protein biomarkers was emphasized. These findings provide insights for the development of clinical protocols that use uEVs for diagnostic purposes.

SNSP113 (PAAG) improves mucociliary transport and lung pathology in the Scnn1b-Tg murine model of CF lung disease.

BACKGROUND: Mucus stasis, a hallmark of muco-obstructive disease, results from impaired mucociliary transport and leads to lung function decline and chronic infection. Although therapeutics that target mucus stasis in the airway, such as hypertonic saline or rhDNAse, show some therapeutic benefit, they do not address the underlying electrostatic defect apparent in mucins in CF and related conditions. We have previously shown poly (acetyl, arginyl) glucosamine (PAAG, developed as SNSP113), a soluble, cationic polymer, significantly improves mucociliary transport in a rat model of CF by normalizing the charge defects of CF mucin. Here, we report efficacy in the CFTR-sufficient, ENaC hyperactive, Scnn1b-Tg mouse model that develops airway muco-obstruction due to sodium hyperabsorption and airway dehydration. METHODS: Scnn1b-Tg mice were treated with either 250 µg/mL SNSP113 or vehicle control (1.38% glycerol in PBS) via nebulization once daily for 7 days and then euthanized for analysis. Micro-Optical Coherence Tomography-based evaluation of excised mouse trachea was used to determine the effect on the functional microanatomy. Tissue analysis was performed by routine histopathology. RESULTS: Nebulized treatment of SNSP113 significantly improved mucociliary transport in the airways of Scnn1b-Tg mice, without altering the airway surface or periciliary liquid layer. In addition, SNSP113 significantly reversed epithelial hypertrophy and goblet cell metaplasia. Finally, SNSP113 significantly ameliorated eosinophilic crystalline pneumonia and lung consolidation in addition to inflammatory macrophage influx in this model. CONCLUSION: Overall, this study extends the efficacy of SNSP113 as a potential therapeutic to alleviate mucus stasis in muco-obstructive diseases in CF and potentially in related conditions.

Alpha-2 macroglobulin for the treatment of neuroma pain in the stump of a below-knee amputee patient.

This case report describes the successful treatment of neuroma pain in the setting of below knee amputations using alpha-2-macroglobulin (A2M). A 34-year-old female patient presented with 9 months of stump pain despite conservative treatment. The exam revealed persistent pain through rest periods and weight-bearing status during therapy. Ultrasound showed neuroma formation with neovascularization. The patient underwent two A2M hydrodissection treatments, 2 weeks apart. The patient reported significant pain relief. Ultrasound showed decreases in neovascularization and cross-sectional area of the neuroma. The patient was able to ambulate pain-free for 2 years and reported no pain since. A2M may be a treatment for patients with neuroma pain in the setting of amputations.

Simplified α2-macroglobulin as a TNF-α inhibitor for inflammation alleviation in osteoarthritis and myocardial infarction therapy.

Tumor necrosis factor α (TNF-α) is a leading proinflammatory cytokine as the master regulator of inflammation in chronic inflammation diseases. Although TNF-α antagonists such as small molecules and peptides are in development, comparable effectiveness in TNF-α neutralization is hardly achieved only with TNF-α capture. In this study, simplified α2-macroglobulin (SM) as a novel TNF-α inhibitor was fabricated to relieve inflammation response by TNF-α capture and internalization with lysosomal degradation. SM was prepared by conjugating a TNF-α-targeting peptide with a receptor binding domain (RBD) derived from α2-macroglobulin through a synthetic biology strategy. SM exhibited effective capture and bioactivity inhibition of TNF-α. Improved endocytosis of TNF-α into lysosomes was observed with SM in macrophages. Even challenged with LPS/IFNγ, the macrophages showed relieved inflammation response with SM treatment. When administrated in chronic inflammation injury in vivo, SM achieved comparable therapeutic efficacy with Infliximab, showing ameliorated cartilage degeneration with relieved inflammation in osteoarthritis (OA) and preserved cardiac function with mitigated myocardium injury in myocardial infarction (MI). These results suggest that SM functioning in TNF-α capture-internalization mechanism might be promising therapeutic alternatives of TNF-α antibodies.

A2M Serves as Promising Biomarker for Chronic Obstructive Pulmonary Disease.

Background: Chronic obstructive pulmonary disease (COPD) often associated with cigarette smoking. However, increasing evidence suggests that non-smoking COPD is much higher than previously thought. This study aims to identify a nonsmoking COPD biomarker and examined its value in diagnosis and prediction of acute exacerbation. Methods: A total of 35 stable COPD patients, 70 acute exacerbation chronic obstructive pulmonary disease (AECOPD) patients and 35 healthy control subjects were included. Plasma α 2 macroglobulin (A2M) and matrix metalloproteinase-9 (MMP-9) levels were measured using the enzyme-linked immunosorbent assay (ELISA) method on all participants. Their association with clinical characteristics and lung function parameters were determined by regression analysis. Receiver operating characteristic (ROC) curve was used to determine the diagnostic sensitivity and specificity. Correlation coefficients were evaluated using Pearson's correlation. Results: Plasma A2M concentration was decreased and MMP-9 concentration, MMP-9/A2M ratio were elevated in stable COPD patients compared with control groups. And MMP-9 expression was significantly higher in AECOPD patients. A2M level was increased in AECOPD patients with infection compared with those without. In addition, there was no statistical difference in A2M levels between smokers and nonsmokers COPD or healthy control subjects. Furthermore, A2M, MMP-9 and MMP-9/A2M were correlated with forced expiratory volume in one second (FEV1)%, FEV1/ forced vital capacity (FVC), CAT and mMRC score in COPD patients, but had no correlation with fraction of exhaled nitric oxide (FeNO) and concentration of alveolar nitric oxide (CaNO). Conclusion: A2M is altered in peripheral blood of COPD patients and correlated with severity and infection. Moreover, there was no significant correlation between the change in A2M and smoking, FeNO and CaNO, suggesting A2M may reflect the overall rather than local inflammation in COPD patients and serve as a potential biomarker for nonsmoking COPD patients.

Exploring the interaction of myricetin with human alpha-2-macroglobulin: biophysical and in-silico analysis.

Myricetin (MYR) is a bioactive secondary metabolite found in plants that is recognized for its nutraceutical value and is an essential constituent of various foods and beverages. It is reported to exhibit a plethora of activities, including antioxidant, antimicrobial, antidiabetic, anticancer, and anti-inflammatory. Alpha-2-macroglobulin (α2M) is a major plasma anti-proteinase that can inhibit proteinases of both human and non-human origin, regardless of their specificity and catalytic mechanism. Here, we explored the interaction of MYR-α2M using various biochemical and biophysical techniques. It was found that the interaction of MYR brings subtle change in its anti-proteolytic potential and thereby alters its structure and function, as can be seen from absorbance and fluorescence spectroscopy. UV spectroscopy of α2M in presence of MYR indicated the occurrence of hyperchromism, suggesting complex formation. Fluorescence spectroscopy reveals that MYR reduces the fluorescence intensity of native α2M with a shift in the wavelength maxima. At 318.15 K, MYR binds to α2M with a binding constant of 2.4 × 103 M-1, which indicates significant binding. The ΔG value was found to be - 7.56 kcal mol-1 at 298.15 K, suggesting the interaction to be spontaneous and thermodynamically favorable. The secondary structure of α2M does not involve any major change as was confirmed by CD analysis. The molecular docking indicates that Asp-146, Ser-172, Glu-174, and Tyr-180 were the key residues involved in α2M-MYR complex formation. This study contributes to our understanding of the function and mechanism of protein and flavonoid binding by providing a molecular basis of the interaction between MYR and α2M.

Retinal Pigment Epithelium-Secreted VEGF-A Induces Alpha-2-Macroglobulin Expression in Endothelial Cells.

Alpha-2-macroglobulin (A2M) is a protease inhibitor that regulates extracellular matrix (ECM) stability and turnover. Here, we show that A2M is expressed by endothelial cells (ECs) from human eye choroid. We demonstrate that retinal pigment epithelium (RPE)-conditioned medium induces A2M expression specifically in ECs. Experiments using chemical inhibitors, blocking antibodies, and recombinant proteins revealed a key role of VEGF-A in RPE-mediated A2M induction in ECs. Furthermore, incubation of ECs with RPE-conditioned medium reduces matrix metalloproteinase-2 gelatinase activity of culture supernatants, which is partially restored after A2M knockdown in ECs. We propose that dysfunctional RPE or choroidal blood vessels, as observed in retinal diseases such as age-related macular degeneration, may disrupt the crosstalk mechanism we describe here leading to alterations in the homeostasis of choroidal ECM, Bruch's membrane and visual function.

Increased serum level of alpha-2 macroglobulin and its production by B-lymphocytes in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), the most common adult's leukemia in the western world, is caused in 95% of the cases by uncontrolled proliferation of monoclonal B-lymphocytes. The complement system in CLL is chronically activated at a low level via the classical pathway (CP). This chronic activation is induced by IgG-hexamers, which are formed after binding to alpha-2-macroglobulin (A2M). The study investigated for the first time the serum levels of A2M in CLL patients, their association with the disease severity, and A2M production by the malignant B-lymphocytes. Blood samples were collected from 65 CLL patients and 30 normal controls (NC) subjects, and used for quantifications of the A2M levels, the complement activation marker (sC5b-9), the complement components C2, C3 and C4, and clinical biochemistry and hematology parameters. The production of A2M was studied in B-lymphocytes isolated from blood samples as well as in CLL and non-CLL cell lines.The serum A2M levels were significantly higher in CLL patients vs NCs, showing values of 3.62 ± 0.22 and 1.97 ± 0.10 mg/ml, respectively. Within the CLL group, A2M levels correlated significantly with the disease stage, with sC5b-9, and with clinical indicators of the disease severity. Increased A2M production was showed in three out of four CLL B-lymphocytic lines that were studied, as compared to non-CLL lines, to a non-lymphocytic line, and to blood-derived primary B-lymphocytes. A2M production was further increased both in primary cells and in the CLL cell-line after incubation with CLL sera, compared to NC sera. This study shows for the first time that serum A2M levels in CLL are significantly increased, likely due to A2M production by the malignant B-lymphocytes, and are correlated with the disease severity and with chronic complement activation. The moderate change in A2M production after incubation with NC sera in-vitro supports the hypothesis that inhibition of excess A2M production can be achieved, and that this may potentially down-regulate the IgG-hexamerization and the resulting chronic CP activation. This may also help restore complement system activity, and eventually improve complement activity and immunotherapy outcomes in CLL.

Antagonism of cysteinyl leukotriene receptors by zafirlukast modulated acute inflammatory reaction in tilapia, Oreochromis niloticus.

To identify activation pathways and effector mechanisms of innate immunity in fish has become relevant for the sanitary management of intensive fish farming. However, little is known about the blocking of cysteinyl leukotrienes receptors (CysLTRs) and their effects in teleost fish. Our study evaluated the anti-inflammatory effect of 250 and 500 µg zafirlukast (antagonist of CysLTRs)/kg b.w., administered orally in the diet, during acute inflammatory reaction induced by Aeromonas hydrophila bacterins in Oreochromis niloticus. 80 tilapia were distributed in 10 aquariums (100L of water each, n = 8) to constitute three treatments: Control (inoculated with A. hydrophila bacterin and untreated); Treated with 250 µg or 500 µg of zafirlukast/kg b.w. and inoculated. To be evaluated in three periods: 6, 24 and 48 h post-inoculation (HPI), totaling nine aquariums. A tenth group was sampled without any stimulus to constitute reference values (Physiological standards). Tilapia treated with zafirlukast demonstrated dose-response effect in the decrease of accumulated inflammatory cells, strongly influenced by granulocytes and macrophages. Zafirlukast treated-tilapia showed decrease in blood leukocyte counts (mainly neutrophils, and monocytes) and reactive oxygen species production. Treatment with zafirlukast resulted in down-regulation of ceruloplasmin, complement 3, alpha2-macroglobulin, transferrin and apolipoprotein A1, as well as up-regulation of haptoglobin. Our study provided convincing results in the pathophysiology of tilapia inflammatory reaction, considering that treatment with zafirlukast, antagonist of cysteinyl leukotriene receptors, resulted in a dose-response effect by suppressing the dynamics between leukocytes in the bloodstream and cell accumulation in the inflamed focus, as well as modulated the leukocyte oxidative burst and the acute phase protein response.

Management of Complications in 257 Cases of Breast Augmentation with Polyacrylamide Hydrogel, using Two Different Strategies: A Retrospective Study.

BACKGROUND: Polyacrylamide hydrogel (PAAG) has been used globally for breast augmentation, leading to long-term clinical complications. However, whether the infiltrated fibrotic capsule should be removed with PAAG to alleviate the complications remains unclear. This study aimed to ascertain different causes of complications and proper management strategies for PAAG removal in augmented breasts. METHODS: From July 2015 to December 2019, patients who underwent breast augmentation with PAAG and in whom surgical intervention was undertaken for PAAG-associated adverse events at Shanghai Ninth People's Hospital were retrospectively reviewed. Patients were categorized into two groups according to whether the fibrotic capsule was removed (RFC) or not (NRFC). Aesthetic outcomes, PAAG residues, and adverse events were evaluated post-operatively to assess whether important issues pertaining to these arose following fibrotic capsule removal. Tissue histology and PAAG degradation analysis were implemented to investigate immune response, degradability, and toxicity of PAAG. RESULTS: Altogether, 257 patients (88 RFC and 169 NRFC patients) were enrolled. 73.4% and 79.5% of the RFC and NRFC groups showed fairly good outcomes, with no significant difference, respectively. (X2 = 0.0804, p = 0.79) Significant differences were found between two surgical techniques upon patient satisfaction, respectively. (X2 = 3.529; p = 0.0301). Predictor of poor outcomes identified scar (OR, 4.555, p = 0.0019) and PAAG residue (OR, 5.379, p = 0.0003). Predictor of patient satisfaction identified post-operative outcomes (OR, 3.797; 95% CI, 1.860-8.923; p = 0.0002) and surgical technique (NRFC) (OR, 2.519; 95% CI, 1.449-4.434; p = 0.0008). CONCLUSIONS: Both treatment strategies showed good results in our study. Removal of the fibrotic capsule from infiltration of PAAG largely depends on the individual psychological condition, aesthetic expectations, complications, and magnetic resonance imaging results. While PAAG does not degrade in the host's body over time, it may elicit immune reactions and chronic inflammation in the long term. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine Ratings, please refer to Table of Contents or online Instructions to Authors www.springer.com/00266.

Universal Accessible Biomarkers of Drug-Induced Tissue Injury and Systemic Inflammation in Rat: Performance Assessment of TIMP-1, A2M, AGP, NGAL, and Albumin.

The ability to monitor for general drug-induced tissue injury (DITI) or systemic inflammation in any tissue using blood-based accessible biomarkers would provide a valuable tool in early exploratory animal studies to understand potential drug liabilities. Here we describe the evaluation of 4 biomarkers of tissue remodeling and inflammation (α2-macroglobulin [A2M], α1-acid glycoprotein [AGP], neutrophil gelatinase-associated lipocalin [NGAL], and tissue inhibitor of metalloproteinases [TIMP-1]) as well as the traditional serum parameter albumin as potential blood-based biomarkers of DITI and systemic inflammatory response (SIR). Biomarker performance was assessed in 51 short-term rat in vivo studies with various end-organ toxicities or SIR and receiver operating characteristic curves were generated to compare relative performances. All 4 biomarkers performed well in their ability to detect DITI and SIR with an area under the curve (AUC) of 0.82-0.78, however TIMP-1 achieved the best sensitivity (at 95% specificity) of 61%; AGP, NGAL, and A2M sensitivity was 51%-52%. AUC for albumin was 0.72 with sensitivity of 39%. A2M was the best performer in studies with only SIR (AUC 0.91). In the subset of studies with drug-induced vascular injury, TIMP-1 performed best with an AUC of 0.96. Poor performance of all tested biomarkers was observed in samples with CNS toxicity. In summary, TIMP-1, A2M, AGP, and NGAL demonstrated performance as sensitive accessible biomarkers of DITI and SIR, supporting their potential application as universal accessible tissue toxicity biomarkers to quickly identify dose levels associated with drug-induced injury in early exploratory rat safety and other studies.

Characterization of the binding between anti-tumor drug 5-fluorouracil and human alpha-2-macroglobulin: spectroscopic and molecular docking analyses.

5-Fluorouracil (5-FU) is a well-recognized anticancer drug used in the treatment of tumors of head, neck and breast. Drug pharmacokinetics is affected upon binding with protein, thus, making drug-protein interactions imperative to study. Present work investigates the interaction between 5-FU and human major antiproteinase-alpha-2-macroglobulin (α2M) by multi-spectroscopic, calorimetric and molecular docking techniques. UV/Visible absorption, intrinsic fluorescence and circular dichroism (CD) spectroscopic methods have been employed to unveil the mode and mechanism of 5-FU-α2M interaction. Synchronous fluorescence showed alteration in the microenvironment of tryptophan and tyrosine residues of protein. Far UV-CD spectra suggest slight alterations in the secondary structure of α2M by 5-FU. Thermodynamic parameters determined by fluorescence quenching experiments and isothermal titration calorimetry (ITC) suggested the involvement of hydrogen bonds and hydrophobic interactions. Moreover, ITC corroborate the spontaneous and exothermic nature of the interaction process. Molecular docking illustrates that 5-FU binds with moderate affinity and Asp953, Tyr1264, Lys1236, Thr1232, Tyr1323 and Leu951 were the main residues involved. Molecular dynamics simulation studies suggested that 5-FU was stabilizing the α2M structure and forming a stable complex. It was concluded that 5-FU lower the antiproteolytic activity of α2M significantly and causes disruption in the native structure and conformation of α2M.Communicated by Ramaswamy H. Sarma.

Comprehensive insight into the molecular interaction of an anticancer drug-ifosfamide with human alpha-2-macroglobulin: biophysical and in silico studies.

Ifosfamide is an active alkylating chemotherapeutic drug chemically related to nitrogen mustard. The pharmacokinetics of drugs is affected upon binding with protein, making the studies on drug-protein interaction promising. The present study investigates the interaction between ifosfamide and human antiproteinase-alpha-2-macroglobulin (α2M) by using multi-spectroscopic and in silico techniques. The UV-visible absorption, intrinsic fluorescence and circular dichroism (CD) spectroscopic methods were employed to unveil the mode and mechanism of ifosfamide-α2M interaction. Fluorescence quenching studies performed at three different temperatures indicated that ifosfamide-α2M complex formation involves static quenching. Far UV-CD spectra revealed a minor alteration in the secondary structure of α2M instigated by ifosfamide. The thermodynamic parameters determined by fluorescence quenching experiment and isothermal titration calorimetry (ITC) suggested that the complex between ifosfamide and α2M involves hydrogen bonding and hydrophobic interactions. Molecular docking illustrates that ifosfamide binds with moderate affinity to Lys1240, Asn173, Ser957, Leu955, Asp953, Lys1216 and Thr1236 residues during the interaction. Molecular dynamic (MD) simulation suggested that the ifosfamide forms a stable complex with α2M. Communicated by Ramaswamy H. Sarma.

Alpha-2-Macroglobulin in Inflammation, Immunity and Infections.

Alpha-2-macroglobulin is an extracellular macromolecule mainly known for its role as a broad-spectrum protease inhibitor. By presenting itself as an optimal substrate for endopeptidases of all catalytic types, alpha-2-macroglobulin lures active proteases into its molecular cage and subsequently 'flags' their complex for elimination. In addition to its role as a regulator of extracellular proteolysis, alpha-2-macroglobulin also has other functions such as switching proteolysis towards small substrates, facilitating cell migration and the binding of cytokines, growth factors and damaged extracellular proteins. These functions appear particularly important in the context of immune-cell function. In this review manuscript, we provide an overview of all functions of alpha-2-macroglobulin and place these in the context of inflammation, immunity and infections.

Induced forms of α2-macroglobulin neutralize heparin and direct oral anticoagulant effects.

Alpha2-macroglobulin (α2M) is a physiological macromolecule that facilitates the clearance of many proteinases, cytokines and growth factors in human. Here, we explored the effect of induced forms of α2M on anticoagulant drugs. Gla-domainless factor Xa (GDFXa) and methylamine (MA)-induced α2M were prepared and characterized by electrophoresis, immunonephelometry, chromogenic, clot waveform and rotational thromboelastometry assays. Samples from healthy volunteers and anticoagulated patients were included. In vivo neutralization of anticoagulants was evaluated in C57Bl/6JRj mouse bleeding-model. Anticoagulant binding sites on induced α2M were depicted by computer-aided energy minimization modeling. GDFXa-induced α2M neutralized dabigatran and heparins in plasma and whole blood. In mice, a single IV dose of GDFXa-induced α2M following anticoagulant administration significantly reduced blood loss and bleeding time. Being far easier to prepare, we investigated the efficacy of MA-induced α2M. It neutralized rivaroxaban, apixaban, dabigatran and heparins in spiked samples in a concentration-dependent manner and in samples from treated patients. Molecular docking analysis evidenced the ability of MA-induced α2M to bind non-covalently these compounds via some deeply buried binding sites. Induced forms of α2M have the potential to neutralize direct oral anticoagulants and heparins, and might be developed as a universal antidote in case of major bleeding or urgent surgery.