Indirect comparison of tisagenlecleucel and historical treatments for relapsed/refractory diffuse large B-cell lymphoma.

No head-to-head trials have compared the efficacy of tisagenlecleucel vs historical treatments for adults with relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). This study indirectly compared the overall survival (OS) and overall response rate (ORR) associated with tisagenlecleucel, using data from the JULIET study (Study of Efficacy and Safety of CTL019 in Adult DLBCL Patients; #NCT02445248), vs historical treatments assessed in the CORAL (Collaborative Trial in Relapsed Aggressive Lymphoma) study follow-up population. To assess treatment effects in the treated (full analysis set [FAS]) and enrolled (intention-to-treat [ITT]) study populations, the JULIET FAS vs the CORAL follow-up FAS and JULIET ITT vs CORAL follow-up ITT populations were separately compared. Propensity score weighting using standardized mortality ratio weight (SMRW) and fine stratification weight (FSW) was used to compare OS and ORR, adjusting for baseline confounders. The results indicated that tisagenlecleucel was associated with a lower hazard of death among the FAS (adjusted hazard ratio [95% confidence interval], both FSW and SMRW, 0.44 [0.32, 0.59]) and ITT populations (FSW, 0.60 [0.44, 0.77]; SMRW, 0.57 [0.44, 0.73]; all, P < .001). Median OS was 12.48 months (JULIET) vs 4.34 to 4.40 months (CORAL) for the FAS, and 8.25 (JULIET) months vs 4.04 to 4.86 (CORAL) months for the ITT populations. Tisagenlecleucel was associated with a significantly higher ORR compared with historical treatments among the FAS (adjusted response rate difference [95% confidence interval], both FSW and SMRW, 36% [22%, 0.48%]; P < .001) and among the ITT populations after SMRW adjustment (11% [0%, 22%]; P = .043). This analysis supports that improved response and OS are achieved in patients with r/r DLBCL treated with tisagenlecleucel compared with those treated with alternative historical treatments.

Prevalence of iron deficiency in pregnant women: A prospective cross-sectional Austrian study.

The aim of the study was to determine, for the first time, in a prospective cross-sectional multicenter study, the prevalence of iron deficiency (ID) in an Austrian pregnant population. A cohort of 425 pregnant women was classified into four groups of different weeks of gestation. Group 1 was monitored longitudinally, while groups 2-4, iron status, were sampled only once. Evaluation of the prevalence of ID was performed by comparing the diagnostic criteria of the WHO to the cutoff proposed by Achebe MM and Gafter-Gvili A (Achebe) and the Austrian Nutrition Report (ANR). In comparison with the ANR, the prevalence of ID was lower in group 1 and higher in groups 2-4 (17.2% vs. 12.17%, 25.84%, 35.29%, and 41.76%, respectively) (p-values < .01 except group 1). According to WHO, the prevalence in group 1 was 12.17% at inclusion, 2 months later 31.7%, and further 2 months later 65.71%, respectively. According to Achebe, the number of cases doubled; for group 1, the number of cases rose from 13 to 42 (115 patients total); for groups 2-4, we observed an increase from 112 to 230 (340 patients total). This study reported a prevalence of around 12% at the beginning of pregnancy, which increased during pregnancy up to 65%. ID can have a massive impact on quality of life, justifying screening, as iron deficiency would be easy to diagnose and treat.

Evaluation of Integrated HPV DNA as Individualized Biomarkers for the Detection of Recurrent CIN2/3 during Post-Treatment Surveillance.

PURPOSE: Post-treatment follow-up in women with cervical pre-cancers (CIN3) is mandatory due to relapse in up to 10% of patients. Standard follow-up based on hrHPV-DNA/cytology co-testing has high sensitivity but limited specificity. The aim of our prospective, multicenter, observational study was to test the hypothesis that an individualized viral-cellular-junction test (vcj-PCR) combined with cytology has a lower false positive rate for the prediction of recurrence compared to standard co-testing. METHODS: Pre-surgical cervical swabs served for the identification of HPV16/18 DNA integration sites by next-generation-sequencing (NGS). Samples taken at 6, 12 and 24 months post-surgery were evaluated by cytology, hrHPV-DNA and the patients' individual HPV-integration sites (vcj-PCR on the basis of NGS). RESULTS: Integration sites were detected in 48 of 445 patients (10.8%), 39 of them had valid follow-up data. The false positive rate was 18.2% (95% CI 8.6-34.4%) for standard hrHPV/cytology at six months compared to 12.1% (95% CI 4.8-27.3%) for vcj-PCR/cytology, respectively (McNemar p = 0.50). Six patients developed recurrences (1 CIN2, 5 CIN3) during follow-up. Standard co-testing detected all, whereas vcj-PCR/cytology detected only five patients with recurrences. Data of 269 patients without evidence of HPV16/18 integration were subject to post-hoc analyses. Standard co-testing revealed a false positive rate of 15.7% (95% CI 11.7-20.7%) and predicted ten of fourteen recurrences at six months. CONCLUSIONS: Although highly specific on its own vcj-PCR could not detect all recurrent CIN2/3. Possible reasons for this unexpected result may be multifocal lesions, intratumoral heterogeneity with respect to HPV integration and/or incident CIN.

Effect of Physical and Chemical Hair Removal Methods on Skin Barrier Function in vitro: Consequences for a Hydrophilic Model Permeant.

BACKGROUND: Although very common in our society, the effect of hair removal on physiological skin parameters and on the ingress of applied chemicals has not been systematically investigated. Thus, as a first step, the aim of the present study was to elucidate the effect of hair removal through epilation (electric epilation, waxing) and depilation (dry and wet shaving, depilatory cream) on skin properties in vitro using the porcine ear model. METHODS: Attenuated total reflection Fourier transform infrared spectroscopy, measurement of the transepidermal water loss (TEWL), visualization by capacitance-based contact imaging, confocal Raman spectroscopy (CRS), diffusion cell studies and tape stripping experiments were employed. RESULTS: Increased TEWL and altered skin permittivity maps were observed. Decreased stratum corneum thickness was observed after waxing. Diffusion cell studies showed increased skin permeation especially in case of dry shaving, electric epilation and waxing. CONCLUSION: Considering CRS and diffusion cell data, a moderate if significant decrease in skin barrier function was found after hair removal by dry shaving (physical skin/material interaction) and epilation methods (plucking out the entire hair, for example, by electrical epilation and waxing). Subsequent experiments will include testing of different permeants covering a broad range of physicochemical properties in vitro and confirming our findings in vivo.

Differences between evidence-based recommendations and actual clinical practice regarding tocolysis: a prospective multicenter registry study.

BACKGROUND: International guidelines recommend that tocolytic therapy be restricted to a single 48-h application. However, multiple cycles of tocolytic therapy and maintenance therapy that exceeds 48 h appear to play a role in daily clinical practice. We aimed to evaluate current trends in clinical practice with respect to treatment with tocolytic agents and to identify differences between evidence-based recommendations and daily clinical practice in Austria. METHODS: A prospective multicenter registry study was conducted from October 2013 through April 2015 in ten obstetrical departments in Austria. Women ≥18 years of age who received tocolytic therapy following a diagnosis of threatened preterm birth were included, and details were obtained regarding clinical characteristics, tocolytic therapy, and pregnancy outcome. RESULTS: A total of 309 women were included. We observed a median of 2 cycles of tocolytic therapy per patient (IQR 1-3) with a median duration of 2 days per cycle (IQR 2-5). Repeat tocolysis was administered in 41.7% of women, resulting in up to six tocolysis cycles; moreover, 40.8% of the first tocolysis cycles were maintenance tocolysis (i.e., longer than 48 h). Only 25.6% of women received one single 48-h tocolysis cycle in which they received antenatal corticosteroids for fetal lung maturation in accordance evidence-based recommendations. CONCLUSIONS: Here, we report a clear disparity between evidence-based recommendations and daily practice with respect to tocolysis. We believe that the general practice of prescribing tocolytic therapy must be revisited. Furthermore, our findings highlight the need for contemporary studies designed to investigate the effectiveness of performing maintenance and/or repetitive tocolysis treatment.

Novel Surface Coatings Using Oxidized Glycosaminoglycans as Delivery Systems of Bone Morphogenetic Protein 2 (BMP-2) for Bone Regeneration.

Tissue engineering of bone requires the delivery of growth factors in a localized, sustained manner. Here, chitosan is used as polycation, while heparin and chondroitin sulfate are employed either as native or oxidized polyanions for formation of multilayers by layer-by-layer technique. The use of oxidized heparin and oxidized chondroitin sulfate permits additional stabilization by cross-linking through imine bond formation between amino groups of polycations and aldehydes of oxidized glycosaminoglycans (oGAGs). Since these multilayers are highly hydrophilic, adhesion of C2C12 myoblasts is improved either by the use of a specific 4 + 9 pH regime with native glycosaminoglycans or a terminal collagen I layer in case of oGAGs. Adhesion and proliferation studies with C2C12 myoblasts, seeded either on bone morphogenetic protein (BMP-2) loaded or non-loaded multilayers, show that intrinsic cross-linking in oGAG-based multilayers supports cell adhesion, spreading, proliferation, and subsequent cell differentiation into osteoblasts. This is related to higher thickness and roughness of multilayers made of oGAGs compared to their native counterparts studied by ellipsometry and atomic force microscopy. Taken together, oGAG multilayer systems provide stable surface coatings and are useful as biocompatible reservoirs for sustained release of BMP-2, paving the way for coating implants and scaffolds for repair and regeneration of bone.

Viral-Cellular DNA Junctions as Molecular Markers for Assessing Intra-Tumor Heterogeneity in Cervical Cancer and for the Detection of Circulating Tumor DNA.

The development of cervical cancer is frequently accompanied by the integration of human papillomaviruses (HPV) DNA into the host genome. Viral-cellular junction sequences, which arise in consequence, are highly tumor specific. By using these fragments as markers for tumor cell origin, we examined cervical cancer clonality in the context of intra-tumor heterogeneity. Moreover, we assessed the potential of these fragments as molecular tumor markers and analyzed their suitability for the detection of circulating tumor DNA in sera of cervical cancer patients. For intra-tumor heterogeneity analyses tumors of 8 patients with up to 5 integration sites per tumor were included. Tumor islands were micro-dissected from cryosections of several tissue blocks representing different regions of the tumor. Each micro-dissected tumor area served as template for a single junction-specific PCR. For the detection of circulating tumor-DNA (ctDNA) junction-specific PCR-assays were applied to sera of 21 patients. Samples were collected preoperatively and during the course of disease. In 7 of 8 tumors the integration site(s) were shown to be homogenously distributed throughout different tumor regions. Only one tumor displayed intra-tumor heterogeneity. In 5 of 21 analyzed preoperative serum samples we specifically detected junction fragments. Junction-based detection of ctDNA was significantly associated with reduced recurrence-free survival. Our study provides evidence that HPV-DNA integration is as an early step in cervical carcinogenesis. Clonality with respect to HPV integration opens new perspectives for the application of viral-cellular junction sites as molecular biomarkers in a clinical setting such as disease monitoring.

Cystine knot growth factors and their functionally versatile proregions.

The cystine knot disulfide pattern has been found to be widespread in nature, since it has been detected in proteins from plants, marine snails, spiders and mammals. Cystine knot proteins are secreted proteins. Their functions range from defense mechanisms as toxins, e.g. ion channel or enzyme inhibitors, to hormones, blood factors and growth factors. Cystine knot proteins can be divided into two superordinate groups. (i) The cystine knot peptides, also referred to - with other non-cystine knot proteins - as knottins, with linear and cyclic polypeptide chains. (ii) The cystine knot growth factor family, which is in the focus of this article. The disulfide ring structure of the cystine knot peptides is made up by the half-cystines 1-4 and 2-5, and the threading disulfide bond is formed by the half-cystines, 3-6. In the growth factor group, the disulfides of half-cystines 1 and 4 pass the ring structure formed by the half-cystines 2-5 and 3-6. In this review, special emphasis will be devoted to the growth factor cystine knot proteins and their proregions. The latter have shifted into the focus of scientific interest as their important biological roles are just to be unravelled.

Conformational stability of the RNP domain controls fibril formation of PABPN1.

The disease oculopharyngeal muscular dystrophy is caused by alanine codon trinucleotide expansions in the N-terminal segment of the nuclear poly(A) binding protein PABPN1. As histochemical features of the disease, intranuclear inclusions of PABPN1 have been reported. Whereas the purified N-terminal domain of PABPN1 forms fibrils in an alanine-dependent way, fibril formation of the full-length protein occurs also in the absence of alanines. Here, we addressed the question whether the stability of the RNP domain or domain swapping within the RNP domain may add to fibril formation. A variant of full-length PABPN1 with a stabilizing disulfide bond at position 185/201 in the RNP domain fibrillized in a redox-sensitive manner suggesting that the integrity of the RNP domain may contribute to fibril formation. Thermodynamic analysis of the isolated wild-type and the disulfide-linked RNP domain showed two state unfolding/refolding characteristics without detectable intermediates. Quantification of the thermodynamic stability of the mutant RNP domain pointed to an inverse correlation between fibril formation of full-length PABPN1 and the stability of the RNP domain.

A core domain of the BMP2 proregion is sufficient for the biogenesis of mature homodimeric growth factor.

The large proregions of cystine knot growth factors exert several important biological functions. Previous results have shown that the proregion of BMP2 retards the activity of the mature growth factor. Hitherto, no information exists about the function of the BMP2 proregion during the biogenesis of the mature growth factor. Here, evidence is presented that the proregion is required for the secretion of BMP2 when present in covalent linkage to the mature domain. In trans complementation, i.e., with the proregion encoded by a separate plasmid, does not result in secretion of homodimeric mature growth factor. An extended variant of a previously in vitro characterized subdomain of the proregion, when being present in covalent linkage to the mature growth factor, restores secretion of mature BMP2, albeit at a reduced level.

Biophysical and structural characterization of a folded core domain within the proregion of growth and differentiation factor-5.

The structure and function(s) of the very large proregions of the transforming growth factor-ß structure family are known in only a few cases. The proregion of growth and differentiation factor (GDF)5 comprises 354 residues. GDF5 therefore belongs to the group of those growth factors with the largest proregions. Here, we report a biophysical analysis of the proform (proGDF5) and the separate proregion. In the absence of the mature part, the proregion folds reversibly to form a monomeric polypeptide that is stabilized by an intramolecular disulfide bond. In the context of the mature part, i.e. in proGDF5, the proregion shows increased thermodynamic stability and contains a higher proportion of secondary structural elements than in its isolated form. A subdomain within the proregion represents a well-folded structure as monitored via biophysical analysis and NMR spectroscopy. Furthermore, two point mutations that are associated with skeletal malformations lead to reduced thermodynamic stability, which is interpreted on the basis of a homology model with the structure of the related latency-associated peptide, representing the proregion of transforming growth factor-ß1.

Molecular analysis of two novel missense mutations in the GDF5 proregion that reduce protein activity and are associated with brachydactyly type C.

Growth and differentiation factor 5 (GDF5) plays a central role in bone and cartilage development by regulating the proliferation and differentiation of chondrogenic tissue. GDF5 is synthesized as a preproprotein. The biological function of the proregion comprising 354 residues is undefined. We identified two families with a heterozygosity for the novel missense mutations p.T201P or p.L263P located in the proregion of GDF5. The patients presented with dominant brachydactyly type C characterized by the shortening of skeletal elements in the distal extremities. Both mutations gave rise to decreased biological activity in in vitro analyses. The variants reduced the GDF5-induced activation of SMAD signaling by the GDF5 receptors BMPR1A and BMPR1B. Ectopic expression in micromass cultures yielded relatively low protein levels of the variants and showed diminished chondrogenic activity as compared to wild-type GDF5. Interestingly, stimulation of micromass cells with recombinant human proGDF5(T201P) and proGDF5(L263P) revealed their reduced chondrogenic potential compared to the wild-type protein. Limited proteolysis of the mutant recombinant proproteins resulted in a fragment pattern profoundly different from wild-type proGDF5. Modeling of a part of the GDF5 proregion into the known three-dimensional structure of TGFß1 latency-associated peptide revealed that the homologous positions of both mutations are conserved regions that may be important for the folding of the mature protein or the assembly of dimeric protein complexes. We hypothesize that the missense mutations p.T201P and p.L263P interfere with the protein structure and thereby reduce the amount of fully processed, biologically active GDF5, finally causing the clinical loss of function phenotype.

Influence of the polypeptide environment next to amyloidogenic peptides on fibril formation.

Alternative folding or fibril formation of proteins is associated with many diseases. Although uncertainty remains for many diseases as to whether the fibrils themselves constitute the main pathogenicity factor, the biophysics or molecular steps leading to fibrils cannot easily be reduced to a common denominator. To date, it is known that fibrils can form (i) upon aberrant (over-)production or false processing, (ii) upon infection with prions that act as seeds and induce unfolding of a thus far native protein--as has been shockingly experienced during the bovine spongiform encephalopathy episode, (iii) when mutations are present that increase the propensity of an otherwise stable protein to aggregate, or (iv) when mutation decreases the overall stability of an individual protein. This review intends to highlight some of the biochemical and biophysical mechanisms that favor fibril formation. Special emphasis is given on the relevance of the polypeptide environment of amyloidogenic segments and the currently discussed driving forces of fibril formation.

Feasibility of newborn screening for guanidinoacetate methyltransferase (GAMT) deficiency.

Guanidinoacetate methyltransferase (GAMT) deficiency causes brain creatine deficiency characterized by developmental delays, speech delay, seizures and autism-like behavior. Identification and therapy at birth because of a positive family history has prevented intellectual disability and seizures in all cases reported. The objective of this study was to develop a method to identify patients with GAMT deficiency from newborn screening blood spots. Creatine and guanidinoacetate were extracted from 10,000 deidentified blood spots using the same protocol routinely used for newborn screening and quantified by stable isotope dilution using deuterated creatine and guanidinoacetate as internal standards. Residual dried blood spots from three infants with GAMT deficiency were used to evaluate the sensitivity of the method. A second tier test using UPLC-MS/MS was performed to analyze samples with a concentration of guanidinoacetate >2.44 µmol/L (99.5th centile of the normal population). Fifty four blood spots required second tier testing in addition to seven blood spots from three patients with GAMT deficiency retrospectively analyzed. With second tier testing, only the samples from GAMT deficiency patients had elevated concentration of guanidinoacetate. Our results show that GAMT deficiency can be identified in newborns using routine extraction methods. The cost of this additional screening is minimal, as it does not require additional instrumentation, procedure, or sample collection. The use of a second tier test can reduce the false positive rate to a minimum. Summary Brain creatine deficiency syndromes cause mental retardation that can be prevented if therapy is initiated early in life. This manuscript reports that infants with GAMT deficiency (one of the brain creatine deficiency syndromes) can be identified from elevated guanidinoacetate in newborn blood spots with virtually absent false-positive results using a second tier test.

Multiplex Identification of Human Papillomavirus 16 DNA Integration Sites in Cervical Carcinomas.

Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3'-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3'-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.

Real-time duplex PCR for simultaneous HPV 16 and HPV 18 DNA quantitation.

HPV 16 and HPV 18 are responsible for more than 75% of cervical cancers and high HPV 16 loads are associated with both prevalent and incident lesions. The objective of the present study was to develop a method allowing the detection and quantitation of HPV 16 and 18 DNA to improve future strategies for cervical cancer screening. A duplex real-time PCR allowing the simultaneous quantitation of both HPV 16 and HPV 18 was carried out. Mixes of HPV 16 and HPV 18 whole genome plasmids were prepared to test a wide range of viral DNA concentrations. The values obtained for each mix of plasmids with the simplex and the duplex PCR were very close to the theoretical values except when a HPV type represented only 1:1000 genome equivalent or lower than the concurrent type. Cervical samples harboring HPV 16, HPV 18 or both types were tested by comparing the results with simplex and duplex real-time PCR assays. HPV 16 and HPV 18 genome titers were similar with the two assays. In conclusion, the real-time duplex PCR proved to be robust for HPV 16 and HPV 18 DNA quantitation.

The unresolved puzzle why alanine extensions cause disease.

The prospective increase in life expectancy will be accompanied by a rise in the number of elderly people who suffer from ill health caused by old age. Many diseases caused by aging are protein misfolding diseases. The molecular mechanisms underlying these disorders receive constant scientific interest. In addition to old age, mutations also cause congenital protein misfolding disorders. Chorea Huntington, one of the most well-known examples, is caused by triplet extensions that can lead to more than 100 glutamines in the N-terminal region of huntingtin, accompanied by huntingtin aggregation. So far, nine disease-associated triplet extensions have also been described for alanine codons. The extensions lead primarily to skeletal malformations. Eight of these proteins represent transcription factors, while the nuclear poly-adenylate binding protein 1, PABPN1, is an RNA binding protein. Additional alanines in PABPN1 lead to the disease oculopharyngeal muscular dystrophy (OPMD). The alanine extension affects the N-terminal domain of the protein, which has been shown to lack tertiary contacts. Biochemical analyses of the N-terminal domain revealed an alanine-dependent fibril formation. However, fibril formation of full-length protein did not recapitulate the findings of the N-terminal domain. Fibril formation of intact PABPN1 was independent of the alanine segment, and the fibrils displayed biochemical properties that were completely different from those of the N-terminal domain. Although intranuclear inclusions have been shown to represent the histochemical hallmark of OPMD, their role in pathogenesis is currently unclear. Several cell culture and animal models have been generated to study the molecular processes involved in OPMD. These studies revealed a number of promising future therapeutic strategies that could one day improve the quality of life for the patients.

Different morphology of amyloid fibrils originating from agitated and non-agitated conditions.

In vitro amyloid formation has been suggested to be a common property of any polypeptide chain depending on particular environmental conditions although in vivo amyloid fibril formation can be promoted by point mutations or triplet expansions. Here, we explored the influence of agitation on fibril formation of amyloidogenic alanine segments fused to Cold Shock Protein B (CspB) of Bacillus subtilis. While without agitation fibril formation was clearly dependent on the presence of an amyloidogenic alanine segment, fibril formation was independent of the amyloidogenic segment under agitation. Agitation even led to fibrillation of native CspB lacking the amyloidogenic segment. Furthermore, agitation not only influenced the kinetics of fibril formation, but also resulted in completely different fibril morphologies. These results indicate that experimental conditions can alter the region that undergoes a conformational change during in vitro fibrillation. Moreover, the data show that deductions from in vitro assays on in vivo fibril formation mechanisms are afflicted with a certain degree of uncertainty and therefore need to be cautiously discussed.

Treatment with bone morphogenetic protein 2 limits infarct size after myocardial infarction in mice.

Various strategies have been devised to reduce the clinical consequences of myocardial infarction, including acute medical care, revascularization, stem cell transplantations, and more recently, prevention of cardiomyocyte cell death. Activation of embryonic signaling pathways is a particularly interesting option to complement these strategies and to improve the functional performance and survival rate of cardiomyocytes. Here, we have concentrated on bone morphogenetic protein 2 (BMP-2), which induces ectopic formation of beating cardiomyocytes during development in the mesoderm and protects neonatal cardiomyocytes from ischemia-reperfusion injury. In a mouse model of acute myocardial infarction, an i.v. injection of BMP-2 reduced infarct size in mice when given after left anterior descending artery ligation. Mice treated with BMP-2 are characterized by a reduced rate of apoptotic cardiomyocytes both in the border zone of the infarcts and in the remote myocardium. In vitro, BMP-2 increases the frequency of spontaneously beating neonatal cardiomyocytes and the contractile performance under electrical pacing at 2 Hz, preserves cellular adenosine triphosphate stores, and decreases the rate of apoptosis despite the increased workload. In addition, BMP-2 specifically induced phosphorylation of Smad1/5/8 proteins and protected adult cardiomyocytes from long-lasting hypoxia-induced cellular damage and oxidative stress without activation of the cardiodepressant transforming growth factor-ß pathway. Our data suggest that BMP-2 treatment may have considerable therapeutic potential in individuals with acute and chronic myocardial ischemia by improving the contractility of cardiomyocytes and preventing cardiomyocyte cell death.

The factor VII-activating protease (FSAP) enhances the activity of bone morphogenetic protein-2 (BMP-2).

Factor VII-activating protease (FSAP) is a circulating protease involved in the pathogenesis of atherosclerosis, calcification, and fibrotic processes. To understand how FSAP controls the balance of local growth factors, we have investigated its effect on the regulation of bone morphogenetic proteins (BMPs). BMP-2 is produced as a large pro-form and secreted as a mature heparin-binding growth factor after intracellular processing by pro-protein convertases (PCs). In this study, we discovered that FSAP enhances the biological activity of mature BMP-2 as well as its pro-form, as shown by osteogenic differentiation of C2C12 myoblasts. These findings were complemented by knockdown of FSAP in hepatocytes, which revealed BMP-2 processing by endogenous FSAP. N-terminal sequencing indicated that pro-BMP-2 was cleaved by FSAP at the canonical PC cleavage site, giving rise to mature BMP-2 (Arg(282)↓Gln(283)), as well as in the N-terminal heparin binding region of mature BMP-2, generating a truncated mature BMP-2 peptide (Arg(289)↓Lys(290)). Similarly, mature BMP-2 was also cleaved to a truncated peptide within its N-terminal region (Arg(289)↓Lys(290)). Plasmin exhibited a similar activity, but it was weaker compared with FSAP. Thrombin, Factor VIIa, Factor Xa, and activated protein C were not effective. These results were further supported by the observation that the mutation of the heparin binding region of BMP-2 inhibited the processing by FSAP but not by PC. Thus, the proteolysis and activation of pro-BMP-2 and mature BMP-2 by FSAP can regulate cell differentiation and calcification in vasculature and may explain why polymorphisms in the gene encoding for FSAP are related to vascular diseases.