Fibrosis occurs when there is an imbalance in extracellular matrix (ECM) deposition and degradation. Excessive ECM deposition results in scarring and thickening of the affected tissue, and interferes ...with tissue and organ homeostasis - mimicking an exaggerated "wound healing" response. Many transforming growth factor-β (TGF-β) ligands are potent drivers of ECM deposition, and additionally, have a natural affinity for the ECM, creating a concentrated pool of pro-fibrotic factors at the site of injury. Consequently, TGF-β ligands are upregulated in many human fibrotic conditions and, as such, are attractive targets for fibrosis therapy. Here, we will discuss the contribution of TGF-β proteins in the pathogenesis of fibrosis, and promising anti-fibrotic approaches that target TGF-β ligands.
Background
Myocardial fibrosis is characterized by excessive cross‐linking and deposition of collagen type I and is involved in left ventricular stiffening and left ventricular diastolic dysfunction ...(LVDD). We investigated whether the effect of spironolactone on LVDD in patients with heart failure with preserved ejection fraction (HFpEF) depends on its effects on collagen cross‐linking and/or deposition.
Methods and results
We investigated 381 HFpEF patients from the multicentre, randomized, placebo‐controlled Aldo‐DHF trial with measures of the E:e' ratio. The ratio of serum carboxy‐terminal telopeptide of collagen type I to serum matrix metalloproteinase‐1 (CITP:MMP‐1, an inverse index of myocardial collagen cross‐linking) and serum carboxy‐terminal propeptide of procollagen type I (PICP, a direct index of myocardial collagen deposition) were determined at baseline and after 1‐year treatment with spironolactone 25 mg once daily or placebo. Patients were classified by CITP:MMP‐1 and PICP tertiles at baseline. While CITP:MMP‐1 tertiles at baseline interacted (P < 0.05) with spironolactone effect on E:e', PICP tertiles did not. In fact, while spironolactone treatment did not modify E:e' in patients with lower CITP:MMP‐1 levels, this ratio was significantly reduced in the remaining spironolactone‐treated patients. In addition, PICP was unchanged in patients with lower CITP:MMP‐1 levels but was reduced in the remaining spironolactone‐treated patients.
Conclusions
A biochemical phenotype of high collagen cross‐linking identifies HFpEF patients resistant to the beneficial effects of spironolactone on LVDD. It is suggested that excessive collagen cross‐linking, which stabilizes collagen type I fibres, diminishes the ability of spironolactone to reduce collagen deposition in these patients.
Duchenne muscular dystrophy (DMD) is a devastating disorder caused by loss of functional dystrophin protein, resulting in muscle wasting. Enhancing muscle growth by inhibiting myostatin, a growth ...factor negatively regulating skeletal muscle mass, is a promising approach to slow disease progression. Direct administration of myostatin propeptide, a natural inhibitor of mature myostatin, has shown limited efficacy probably due to low serum stability. Here, we demonstrate that serum stability, delivery efficiency and efficacy of propeptide can be significantly enhanced by anchoring propeptide to the surface of exosomes by fusing the inhibitory domain of myostatin propeptide into the second extracellular loop of CD63 (EXOpro). Repeated administrations of EXOpro accelerated muscle regeneration and growth, resulting in significantly increased muscle mass and functional rescue without any detectable toxicity in mdx mice. Importantly, EXOpro partially rehabilitated bone structure and promoted bone regeneration in mdx mice. Our findings demonstrate that anchoring to exosomes increased delivery and serum stability of propeptide and augmented the inhibitory efficacy of myostatin propeptide and thus provide a delivery platform for propeptide-based intervention in DMD.
Background
Collagen biomarkers may correlate with incident heart failure (HF) and its subtypes. We hypothesized that circulating procollagen type III N‐terminal propeptide (PIIINP) and collagen type ...I carboxy‐terminal telopeptide (ICTP) predict incident HF.
Methods and Results
We used a stratified sampling design in a multiethnic sample of 3187 subjects, initially aged 45 to 84 years and free of cardiovascular disease. We assayed baseline serum PIIINP and ICTP concentrations using radioimmunoassay. Incident HF was adjudicated, distinguishing reduced ejection fraction (HFrEF; EF <45%) from preserved EF (HFpEF; EF ≥45%). The incidence density for HFpEF and HFrEF was computed using Poisson regression per SD for each of PIIINP and ICTP, adjusting in model 1 for age, race, sex, and renal function or in model 2 for these variables plus blood pressure and medication. Mean (SD) ICTP was 3.38±1.77 μg/L, and mean (SD) PIIINP was 5.48±2.04 μg/L. Among the HF cases, 96 were HFrEF and 107 were HFpEF. Neither ICTP nor PIIINP significantly predicted incident HFrEF. The incidence density for HFpEF per 100 people observed for 13 years was 1.65 for low PIIINP (lower 6 octiles) versus 3.00 for higher PIIINP (P=0.002) in model 1 and correspondingly 1.45 versus 2.59 (P=0.003) in model 2. For low ICTP (lower 7 octiles) versus higher ICTP (octile 8), incidence densities were 1.79 versus 3.64 (P=0.002) in model 1 and 1.58 versus 3.12 (P=0.002) in model 2.
Conclusions
High levels of circulating ICTP and PIIINP as collagen biomarkers appear to be associated with incident HFpEF, but not HFrEF.
The alpha-glucosidase inhibitor acarbose is an antidiabetic drug delaying assimilation of carbohydrates and, thus, increasing the amount of carbohydrates in the distal parts of the intestines, which ...in turn increases circulating levels of the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1). As GLP-1 may suppress bone resorption, acarbose has been proposed to potentiate meal-induced suppression of bone resorption. We investigated the effect of acarbose treatment on postprandial bone resorption in patients with type 2 diabetes and used the GLP-1 receptor antagonist exendin(9-39)NH2 to disclose contributory effect of acarbose-induced GLP-1 secretion.
In a randomised, placebo-controlled, double-blind, crossover study, 15 participants with metformin-treated type 2 diabetes (2 women/13 men, age 71 (57–85 years), BMI 29.7 (23.6–34.6 kg/m2), HbA1c 48 (40–74 mmol/mol)/6.5 (5.8–11.6 %) (median and range)) were subjected to two 14-day treatment periods with acarbose and placebo, respectively, separated by a six-week wash-out period. At the end of each period, circulating bone formation and resorption markers were assessed during two randomised 4-h liquid mixed meal tests (MMT) with infusions of exendin(9-39)NH2 and saline, respectively. Glucagon-like peptide 2 (GLP-2) was also assessed.
Compared to placebo, acarbose impaired the MMT-induced suppression of CTX as assessed by baseline-subtracted area under curve (P = 0.0037) and nadir of CTX (P = 0.0128). During acarbose treatment, exendin(9-39)NH2 infusion lowered nadir of CTX compared to saline (P = 0.0344). Neither parathyroid hormone or the bone formation marker procollagen 1 intact N-terminal propeptide were affected by acarbose or GLP-1 receptor antagonism. Acarbose treatment induced a greater postprandial GLP-2 response than placebo treatment (P = 0.0479) and exendin(9-39)NH2 infusion exacerbated this (P = 0.0002).
In patients with type 2 diabetes, treatment with acarbose reduced postprandial suppression of bone resorption. Acarbose-induced GLP-1 secretion may contribute to this phenomenon as the impairment was partially reversed by GLP-1 receptor antagonism. Also, acarbose-induced reductions in other factors reducing bone resorption, e.g. glucose-dependent insulinotropic polypeptide, may contribute.
•Alpha-glucosidase inhibition increases postprandial glucagon-like peptide 1 (GLP-1) secretion.•This has been suggested to potentiate postprandial suppression of bone resorption during alpha-glucosidase inhibition.•We used a GLP-1 receptor antagonist during alpha-glucosidase inhibitor treatment to elucidate this.•The alpha-glucosidase inhibitor acarbose reduced postprandial suppression of bone resorption.•GLP-1 receptor antagonism reversed acarbose's effect on postprandial bone resorption.
Cocoonase, a protein that is produced by the silkworm (Bombyx mori), is thought to specifically digest the sericin protein of the cocoon and has a high homology with trypsin. Similar to trypsin, ...cocoonase is folded as an inactive precursor protein which is activated by releasing the propeptide moiety. However, the mechanism responsible for the activation of its catalytic structure has not yet been determined in detail. Therefore, to investigate the activation and folding mechanism of cocoonase, recombinant cocoonase (CCN) and prococoonase (proCCN) were over-expressed in E. coli cells.
Both recombinant proteins (proCCN and CCN) were expressed as inclusion bodies in E. coli cells and their folding was examined under several sets of conditions. After the refolding reactions, both of the recombinant proteins were present as the oxidized soluble forms. The proCCN protein was then auto-processed to release the propeptide region for activation. Interestingly, the CCN (CCN∗) derived from the refolded proCCN showed a much stronger protease activity than the refolded CCN from the reduced CCN in a protease assay using Bz-Arg-OEt as a substrate. In addition, the secondary structure of the refolded CCN protein was similar to that of the CCN∗ protein, as evidenced by CD measurements. These results suggest that the CCN protein becomes trapped in a molten globule-like state without the assistance of the propeptide region during the folding process. We therefore conclude that the propeptide region of CCN kinetically accelerates the folding of CCN to adopt the correct conformation of cocoonase at the final step of the folding pathway.
•The in vitro folding of prococoonase (proCCN) and cocoonase (CCN) was examined using the recombinant proteins expressed in E. coli cells.•The mature form, cocoonase, was not able to fold to the final active conformation by itself.•The propeptide sequence of cocoonase is absolutely required for achieving the correct conformation.•The propeptide sequence kinetically accelerates the folding of cocoonase at the final step of the folding pathway.
Summary
Endothelial cell (EC) activation plays a key role in the pathogenesis of pulmonary microvascular occlusion, which is a hallmark of severe coronavirus disease 2019 (COVID‐19). Consistent with ...EC activation, increased plasma von Willebrand factor antigen (VWF:Ag) levels have been reported in COVID‐19. Importantly however, studies in other microangiopathies have shown that plasma VWF propeptide (VWFpp) is a more sensitive and specific measure of acute EC activation. In the present study, we further investigated the nature of EC activation in severe COVID‐19. Markedly increased plasma VWF:Ag median (interquatile range, IQR) 608·8 (531–830)iu/dl and pro‐coagulant factor VIII (FVIII) levels median (IQR) 261·9 (170–315) iu/dl were seen in patients with severe severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection. Sequential testing showed that these elevated VWF–FVIII complex levels remained high for up to 3 weeks. Similarly, plasma VWFpp levels were also markedly elevated median (IQR) 324·6 (267–524) iu/dl. Interestingly however, the VWFpp/VWF:Ag ratio was reduced, demonstrating that decreased VWF clearance contributes to the elevated plasma VWF:Ag levels in severe COVID‐19. Importantly, plasma VWFpp levels also correlated with clinical severity indices including the Sequential Organ Failure Assessment (SOFA) score, Sepsis‐Induced Coagulopathy (SIC) score and the ratio of arterial oxygen partial pressure to fractional inspired oxygen (P/F ratio). Collectively, these findings support the hypothesis that sustained fulminant EC activation is occurring in severe COVID‐19, and further suggest that VWFpp may have a role as a biomarker in this setting.
The disulfide bonds formed in the SAPA domain of a recombinant version of the NH2-terminal propeptide (SP-BN) from the precursor of human pulmonary surfactant protein B (SP-B) were identified through ...sequential digestion of SP-BN with GluC/trypsin or thermolysin/GluC, followed by mass spectrometry (MS) analysis. MS spectra allowed identification of disulfide bonds between Cys32-Cys49 and Cys40-Cys55, and we propose a disulfide connectivity pattern of 1–3 and 2–4 within the SAPA domain, with the Cys residues numbered according to their position from the N-terminus of the propeptide sequence. The peaks with m/z ∼ 2136 and ∼ 1780 in the MS spectrum of the GluC/trypsin digest were assigned to peptides 24AWTTSSLACAQGPE37 and 45QALQCR50 linked by Cys32-Cys49 and 38FWCQSLE44 and 51ALGHCLQE58 linked by Cys40-Cys55 respectively. Tandem mass spectrometry (MS/MS) analysis verified the position of the bonds. The results of the series ions, immonium ions and internal fragment ions were all compatible with the proposed 1–3/2–4 position of the disulfide bonds in the SAPA domain. This X-pattern differs from the kringle-type found in the SAPB domain of the SAPLIP proteins, where the first Cys in the sequence links to the last, the second to the penultimate and the third to the fourth one. Regarding the SAPB domain of the SP-BN propeptide, the MS analysis of both digests identified the bond Cys100-Cys112, numbered 7–8, which is coincident with the bond position in the kringle motif.
The SAPLIP (saposin-like proteins) family encompasses several proteins with homology to saposins (sphingolipids activator proteins). These are proteins with mainly alpha-helical folds, compact packing including well conserved disulfide bonds and ability to interact with phospholipids and membranes. There are two types of saposin-like domains termed as Saposin A (SAPA) and Saposin B (SAPB) domains. While disulfide connectivity has been well established in several SAPB domains, the position of disulfide bonds in SAPA domains is still unknown. The present study approaches a detailed proteomic study to determine disulfide connectivity in the SAPA domain of the precursor of human pulmonary surfactant-associated protein SP-B. This task has been a challenge requiring the combination of different sequential proteolytic treatments followed by MS analysis including MALDI-TOF and tandem mass MS/MS spectrometry. The determination for first time of the position of disulfide bonds in SAPA domains is an important step to understand the structural determinants defining its biological functions.
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•GluC/trypsin double digestion followed by MS and MS/MS determined the disulfide connectivity in a SAPA domain.•A theoretical handy work assigned internal and terminal disulfide linked ions to peak signals in MS/MS spectra.•A pattern 1–3 and 2–4 is found for disulfide bonds in the SAPA domain of the human proSP-B precursor.•The disulphide pattern determined by this SAPA domain differs from the established SAPB kringle domain.
Patients implanted with osseointegrated (OI) prosthetic systems have reported vastly improved upper and lower extremity prosthetic function compared with their previous experience with ...socket‐suspension systems. However, OI systems have been associated with superficial and deep‐bone infections and implant loosening due, in part, to a failure of the osseointegration process. Although monitoring the osseointegration using circulating biomarkers has clinical relevance for understanding the progression of osseointegration with these devices, it has yet to be established. Ten patients were enrolled in this study. Blood samples were collected at pre‐selected times, starting before implantation surgery, and continuing to 12 months after the second surgery. Bone formation markers, bone resorption markers, and circulating amino acids were measured from blood samples. A linear mixed model was generated for each marker, incorporating patient ID and age with the normalized marker value as the response variable. Post hoc comparisons were made between 1 week before Stage 1 Surgery and all subsequent time points for each marker, followed by multiple testing corrections. Serial radiographic imaging of the residual limb containing the implant was obtained during follow‐up, and the cortical index (CI) was calculated for the bone at the porous region of the device. Two markers of bone formation, specifically bone‐specific alkaline phosphatase (Bone‐ALP) and amino‐terminal propeptide of type I procollagen (PINP), exhibited significant increases when compared with the baseline levels of unloaded residual bone prior to the initial surgery, and they subsequently returned to their baseline levels by the 12‐month mark. Patients who experienced clinically robust osseointegration experienced increased cortical bone thickness at the porous coated region of the device. A medium correlation was observed between Bone‐ALP and the porous CI values up to PoS2‐M1 (p = .056), while no correlation was observed for PINP. An increase in bone formation markers and the lack of change observed in bone resorption markers likely reflect increased cortical bone formation induced by the end‐loading design of the Utah OI device used in this study. A more extensive study is required to validate the correlation observed between Bone‐ALP and porous CI values.
Bone turnover: Biology and assessment tools Szulc, Pawel
Best Practice & Research Clinical Endocrinology & Metabolism,
October 2018, 2018-10-00, 20181001, Letnik:
32, Številka:
5
Journal Article
Recenzirano
Bone turnover includes two processes: resorption (removal of old bone) and formation (laying down of new bone). N-terminal propeptide of type I procollagen (PINP) and C-telopeptide of type I collagen ...(CTX-I) are markers of bone formation and resorption, respectively, that the International Osteoporosis Foundation and the International Federation of Clinical Chemistry recommend for clinical use. Bone turnover markers (BTM) are subject to sources of variability, including feeding (lower resorption) and recent fracture (increased levels of all markers). Controllable patient-related factors should be adapted as much as possible (eg blood collection after an overnight fast) to minimize pre-analytical variability. Uncontrollable factors should be considered in the interpretation of the BTM measurements. BTM do not improve prediction of bone loss or fracture within an individual. In osteoporotic patients, BTM may help to assess the response to anabolic and antiresorptive therapies, to assess compliance to the treatment, or to indicate possible secondary causes of osteoporosis. BTM reflect changes in bone metabolism induced by anti-osteoporotic treatment. Anti-resorptive drugs induce a rapid dose-dependent decrease in bone resorption, whereas bone formation stimulating medications increase the levels of bone formations markers. BTM may be used for monitoring anti-osteoporosis therapy. The expected effect during the anti-resorptive therapy is to decrease the PINP by at least 10 ng/mL and to attain the target level of less than 35 ng/mL. The expected effect during the bone formation-stimulating therapy is to increase the PINP by at least 10 ng/mL and to attain the target level of more than 69 ng/mL.
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