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Background: Circulating levels of the antiangiogenic protein, vasoinhibin, derived from the proteolytic cleavage of prolactin (PRL), in prolactinoma are unknown, as is the molecular nature of its isoforms. Dimerization of recombinant vasoinhibin has been reported., Methods: Vasoinhibin in a human serum sample was identified by using preparative electrophoresis with subsequent SDS-PAGE and Western blot analysis, as well as mass spectrometry (MS) and ELISA., Results: MS identified a partial vasoinhibin sequence in a 14-kDa protein band from human serum, which eluted in the 28-kDa fraction from the preparative electrophoresis. Measurement of vasoinhibin levels by ELISA identified a concentration of 284 ng/mL at a PRL level of 9,850 ng/mL. Recombinant human vasoinhibin demonstrated dimerization and multimerization when analyzed directly by SDS-PAGE and Western blot analysis under reducing and non-reducing conditions, as well as after immunoprecipitation., Conclusions: The vasoinhibin sequence was identified in a higher molecular weight fraction, corroborating experi-mental evidence showing the dimerization and aggregation of recombinant human vasoinhibin. This report is sig-nificant, regarding the higher risk of cardiovascular disease and mortality in male patients with hyperprolactin-emia as well as emerging reports of linking PRL and vasoinhibin levels in patients with prolactinoma with left ventricular dysfunction and Takotsubo syndrome.

Background: The evaluation of low abundance biomarkers in the circulating low molecular weight serum proteome is an important source of information. Techniques for sample preparation to remove high abundant proteins and to enrich the low molecular weight fraction are usually required prior to novel biomarker detection., Methods: A continuous elution electrophoresis was used to separate the low molecular weight serum proteins from the high abundance serum proteins, such as albumin and immunoglobulins. Centrifugal concentration, SDS-PAGE, and total protein staining were performed to analyze eluted protein fractions., Results: Consecutive concentrated serum protein fractions demonstrate separation at a high resolution of 1 - 2 kDa below 20 kDa., Conclusions: Continuous elution electrophoresis is an adequate method to eliminate high abundance proteins which interfere with the detection of low abundance biomarkers in the low molecular weight proteome and to enrich its proteins for subsequent detection and clinical evaluation.

Hormonal factors affecting the vascular adaptions of the uteroplacental unit in noncomplicated and complicated pregnancies are of interest. Here, 4 human placentas from women with and without preeclampsia (PE) were investigated for the presence of placental lactogen (PL)-derived, antiangiogenic vasoinhibin. Western blotting and mass spectrometry of placental tissue revealed the presence of a 9-kDa PL-derived vasoinhibin, the normal 22-kDa full-length PL, and a 28-kDa immunoreactive protein of undetermined nature. The sequence of the 9-kDa vasoinhibin includes the antiangiogenic determinant of vasoinhibin and could constitute a relevant factor in normal pregnancy and PE., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

Vasoinhibin is a protein hormone with antiangiogenic, antivasodilatatory, and antivasopermeability effects generated by the proteolytic cleavage of prolactin. The discovery of its role in diabetic retinopathy and peripartum cardiomyopathy led to the evaluation of new pharmacological treatments in clinical interventional trials. However, the quantitative evaluation of vasoinhibin in biological samples from patients has not been possible due to the lack of vasoinhibin-specific antibodies. Recently, loop 1 of vasoinhibin was identified to have a different three-dimensional structure compared to PRL, and thus to contain vasoinhibin-specific epitopes. Here, we report the development of two sets of vasoinhibin-specific monoclonal antibodies against two neighboring regions of the vasoinhibin loop 1. An experimental sandwich ELISA with two monoclonal anti-vasoinhibin antibodies was developed, which had no cross-reactivity to recombinant human full-length prolactin. The ELISA had a quantitation limit of 100 ng/ml, and intra-assay- and inter-assay coefficients of variation of 12.5% and 14%, respectively. The evaluation of 15 human serum samples demonstrated concentrations of below limit of detection (n=3), below limit of quantitation (n=1) and between 0.23 µg/ml (230 ng/ml) to 605 µg/ml (n=12) in the quantifiable range. Despite the high specificity of the monoclonal-monoclonal antibody sandwiches which discriminate vasoinhibin from PRL, there might be cross-reactivities by serum proteins other than vasoinhibin. A fully established vasoinhibin ELISA may support diagnostic and therapeutic measures in vascular diseases., Competing Interests: The authors declare the following competing interests: CC, JPR, MZ, GME, JT, and TB are inventors of a pending Mexican (MX/E/2019/079075) and International (PCT/EP2020/069154) patent application covering the use of the anti-vasoinhibin monoclonal antibodies for diagnostic purposes. The Universidad Nacional Autónoma de México (UNAM), and the authors JT and TB are owners of the pending-patent applications. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Müller, Robles, Zamora, Ebnet, Markl-Hahn, Martínez de la Escalera, Clapp, Bertsch and Triebel.)