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Background and Objective: Colorectal cancer (CRC) is a major health problem in developed countries. Adenomatous lesions in the large bowel are the main precursors of CRC and the adenoma-adenocarcinoma sequence still provides a solid model for research on carcinogenesis. The finding of local renin-angiotensin systems (RAS) has been crucial to understand the role of this peptidergic system in cancer and has opened new perspectives in the study of colorectal carcinogenetic processes. Methods: In this study we analyzed the immunohistochemical expression of three main RAS receptors (AT1, AT2 and MAS) in a large series of CRC samples (n=161), including uninvolved intestinal mucosa-adenoma-adenocarcinoma sequences from the same patients (n=50). Results: 1) AT1 and AT2 showed a biphasic expression pattern along the sequence. The expression significantly decreased in adenomas with respect to uninvolved mucosa but increased in CRCs. 2) AT2 expression was lower in advanced CRCs with high local invasion (pT4), high stage (IV), high nodal (N2) and vascular invasion. 3) MAS receptor was moderately expressed in the uninvolved mucosa and in adenomas. This expression increased very significantly in CRC tissues. Conclusions: These results suggest that: 1) RAS receptors are differentially regulated as the genetic and epigenetic alterations accumulate throughout the uninvolved mucosa-adenoma-CRC sequence. 2) Loss of AT2 expression could contribute to the aggressive behavior of advanced CRC cells.

Osteoarthritis (OA) is a debilitating condition that significantly impacts its patients and is closely associated with advancing age and senescence. Treatment with autologous platelet rich plasma (PRP) is a novel approach that is increasingly being researched for its effects. Subchondral bone mesenchymal stromal cells (MSCs) are key progenitors that form bone and cartilage lineages that are affected in OA. This study investigated the changes in subchondral bone MSCs before and after combined intraosseous (IO) and intraarticular (IA) PRP infiltration. Patient bone marrow aspirates were collected from 12 patients (four male, eight female) aged 40–86 years old (median 59.5). MSCs were expanded in standard media containing human serum to passage 1 and analysed for their colony-forming potential, senescence status, and gene expression. Hip dysfunction and Osteoarthritis Outcome Score (HOOS) at baseline and 6 months post second infiltration were used to assess the clinical outcomes; seven patients were considered responders and five non-responders. The number of colony-forming MSCs did not increase in the post treatment group, however, they demonstrated significantly higher colony areas (14.5% higher compared to Pre) indicative of enhanced proliferative capacity, especially in older donors (28.2% higher). Senescence assays also suggest that older patients and responders had a higher resistance to senescent cell accumulation. Responder and non-responder MSCs tended to differ in the expression of genes associated with bone formation and cartilage turnover including osteoblast markers, matrix metalloproteinases, and their inhibitors. Taken together, our data show that in hip OA patients, combined IO and IA PRP infiltrations enhanced subchondral MSC proliferative and stress-resistance capacities, particularly in older patients. Future investigation of the potential anti-ageing effect of PRP infiltrations and the use of next-generation sequencing would contribute towards better understanding of the molecular mechanisms associated with OA in MSCs.

One of the most severe effects of coronavirus disease 2019 (COVID-19) is lung disorders such as acute respiratory distress syndrome. In the absence of effective treatments, it is necessary to search for new therapies and therapeutic targets. Platelets play a fundamental role in respiratory disorders resulting from viral infections, being the first line of defense against viruses and essential in maintaining lung function. The direct application of platelet lysate (PL) obtained from the platelet-rich plasma of healthy donors could help in the improvement of the patient due its anti-inflammatory, immunomodulatory, antifibrotic, and repairing effects. This work evaluates PL nebulization by analyzing its levels of growth factors and its biological activity on lung fibroblast cell cultures, besides describing a scientific basis for its use in this kind of pathology. The data of the work suggest that the molecular levels and biological activity of the PL are maintained after nebulization. Airway administration would allow acting directly on the lung tissue modulating inflammation and stimulating reparative processes on key structures such as the alveolocapillary barrier, improving the disease and sequels. The protocol developed in this work is a first step for the study of nebulized PL both in animal experimentation and in clinical trials.

first_page settings Open AccessArticle Effects of Platelet-Rich Plasma on Cellular Populations of the Central Nervous System: The Influence of Donor Age by Diego Delgado 1, Ane Miren Bilbao 2, Maider Beitia 1, Ane Garate 1, Pello Sánchez 1, Imanol González-Burguera 3,4, Amaia Isasti 4,5, Maider López De Jesús 4,5,6, Jone Zuazo-Ibarra 7, Alejandro Montilla 7 [OrcID] , María Domercq 7 [OrcID] , Estibaliz Capetillo-Zarate 7,8, Gontzal García del Caño 3,4 [OrcID] , Joan Sallés 4,5,6, Carlos Matute 7 and Mikel Sánchez 1,2,* 1 Advanced Biological Therapy Unit, Hospital Vithas Vitoria, 01008 Vitoria-Gasteiz, Spain 2 Arthroscopic Surgery Unit, Hospital Vithas Vitoria, 01008 Vitoria-Gasteiz, Spain 3 Department of Neurosciences, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), 01008 Vitoria-Gasteiz, Spain 4 Bioaraba, Neurofarmacología Celular y Molecular, 01008 Vitoria-Gasteiz, Spain 5 Department of Pharmacology, Faculty of Pharmacy, University of the Basque Country (UPV/EHU), 01008 Vitoria-Gasteiz, Spain 6 Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), 28029 Madrid, Spain 7 Achucarro Basque Center for Neuroscience, CIBERNED and Departamento de Neurociencias, Universidad del País Vasco (UPV/EHU), 48940 Leioa, Spain 8 IKERBASQUE, Basque Foundation for Science, 48009 Bilbao, Spain * Author to whom correspondence should be addressed. Academic Editor: Francesca Santilli Int. J. Mol. Sci. 2021, 22(4), 1725; https://doi.org/10.3390/ijms22041725 Received: 24 November 2020 / Revised: 12 January 2021 / Accepted: 3 February 2021 / Published: 9 February 2021 (This article belongs to the Section Molecular Endocrinology and Metabolism) Download PDF Browse Figures Citation Export Abstract Platelet-rich plasma (PRP) is a biologic therapy that promotes healing responses across multiple medical fields, including the central nervous system (CNS). The efficacy of this therapy depends on several factors such as the donor’s health status and age. This work aims to prove the effect of PRP on cellular models of the CNS, considering the differences between PRP from young and elderly donors. Two different PRP pools were prepared from donors 65–85 and 20–25 years old. The cellular and molecular composition of both PRPs were analyzed. Subsequently, the cellular response was evaluated in CNS in vitro models, studying proliferation, neurogenesis, synaptogenesis, and inflammation. While no differences in the cellular composition of PRPs were found, the molecular composition of the Young PRP showed lower levels of inflammatory molecules such as CCL-11, as well as the presence of other factors not found in Aged PRP (GDF-11). Although both PRPs had effects in terms of reducing neural progenitor cell apoptosis, stabilizing neuronal synapses, and decreasing inflammation in the microglia, the effect of the Young PRP was more pronounced. In conclusion, the molecular composition of the PRP, conditioned by the age of the donors, affects the magnitude of the biological response. This work was funded by the Provincial Council of Alava through the AlavaInnova Program, Basque Government through the GAITEK Program, Ministry of Economy, Industry and Competitiveness (CTQ2017-85686-R), Spanish Ministry of Education and Science (PID2019-109724RB-I00), Basque Government (IT1203-19, IT1230-19, and KK-2020/00034) and Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED).

Colorectal cancer (CRC) is the second cause of death in men and the third in women. This work deals with the study of the low molecular weight protein fraction of sera from patients who underwent surgery for CRC and who were followed for several years thereafter. MALDI-TOF MS was used to identify serum peptidome profiles of healthy controls, non-metastatic CRC patients and metastatic CRC patients. A multiple regression model was applied to signals preliminarily selected by SAM analysis to take into account the age and gender differences between the groups. We found that, while a signal m/z 2021.08, corresponding to the C3f fragment of the complement system, appears significantly increased only in serum from metastatic CRC patients, a m/z 1561.72 signal, identified as a prothrombin fragment, has a significantly increased abundance in serum from non-metastatic patients as well. The findings were also validated by a bootstrap resampling procedure. The present results provide the basis for further studies on large cohorts of patients in order to confirm C3f and prothrombin as potential serum biomarkers. Thus, new and non-invasive tests might be developed to improve the classification of colorectal cancer. This work was supported by grants from Italian Ministry of Health (5×1000 Funds 2013 and 2015) and IT8-11/13 from the Basque Government.

Colorectal cancer (CRC) is a major health problem in elderly people because of its high incidence and high mortality rate. Despite early screening programs, more than half of CRC patients are diagnosed at advanced stages. Fibroblast activation protein-alpha (FAP) expression in cancer-associated fibroblasts (CAFs) has been associated with a higher risk of metastases and poor survival. Here, we have analyzed the immunohistochemical expression of FAP in 41 adenoma-carcinoma sequences. In addition, FAP expression was analyzed individually and in combination with beta-catenin (BCAT), CD44 and Cyclin-D1 expression in primary tumors and in their corresponding lymph node and liver metastases (n=294). Finally, soluble FAP (sFAP) levels in plasma from CRC patients (n=127) were also analyzed by ELISA. FAP was expressed only in CRC tissue and its expression level was found to be higher in tumors exhibiting deeper local invasion and poorer cancer cell differentiation. FAP and concomitant nuclear BCAT expression in cancer cells at the infiltrating front of primary tumors and in lymph node metastases was independently associated with 5- and 10-year cancer specific and disease-free survival. Moreover, lower sFAP levels correlated with poorer survival. These findings support the potential importance of FAP as a biomarker of CRC development and progression. This work was partially funded by the ELKARTEK 18/10 grant from the Basque Government.

The biological action of platelet-rich plasma (PRP) could slow down the osteoarthritis progression, resulting in a delay of joint replacement. This work aims to evaluate the ability of PRP to postpone and even avoid knee replacement in patients with knee osteoarthritis (KOA) analyzing, on the one hand, the time of delay and on the other hand the percentage of patients without undergoing total knee arthroplasty (TKA). A retrospective analysis and a survival analysis were conducted. KOA patients who underwent knee replacement between 2014 and 2019 and previously received PRP infiltrations were included in the retrospective analysis. Regarding survival analysis, KOA patients who received PRP treatment during 2014 and with follow-up until 2019 were included. The dates of PRP treatment and TKA, KOA severity, age of the patients, number of PRP cycles, and administration route were analyzed. This work included 1084 patients of which 667 met the inclusion criteria. 74.1% of the patients in the retrospective study achieved a delay in the TKA of more than 1.5 years, with a median delay of 5.3 years. The survival analysis showed that 85.7% of the patients did not undergo TKA during the five year follow-up. The severity degree, age, PRP cycles, and administration route had a statistically significant influence on the efficacy of PRP in delaying surgery. These data suggest that the application of PRP in KOA patients is a treatment that could delay TKA, although further studies are needed to understand and improve this therapy.

Integrity of serum samples is a major concern for biomarker discovery in many fields, including oncology. A gradual degradation of the fibrinopeptide A (fpA) as a result of the preservation process was already observed. In this work, we investigated this process to define a method for the assessment of sera integrity based on the total amount of fpA and its degradation pattern. SeraDeg software was developed. It identifies fpA fragments in spectra and extracts their abundances. The SeraDeg Quality Score (SDQS) is finally determined for each spectrum depending on its total fpA abundance and on the ratio of its higher mass versus lower mass fragments abundances. Spectra were evaluated for the total amount of fpA and the percent contribution of its fragments. The total fpA abundance is significantly lower in cryo-preserved samples than in fresh sera. Moreover, fresh sera present higher percentages of the most intact forms of fpA. Four relevant fpA fragments were identified. We developed and made available on-line SeraDeg, a software able to screen serum spectra, compute fpA related parameters and assign quality scores. SeraDeg was validated using spectra from a set of 250 samples. Biological significance The susceptibility of fpA to degradation suggests its use as quality indicator of cryo-preserved serum samples. SeraDeg can support the adoption of this method which may especially be useful in differential sero-proteomics for wide-range biomarker discovery. Moreover, it discloses the doors to a critical re-evaluation of previous experiments in light of the quality of serum samples as assessed by fpA degradation analysis.

Rennin-angiotensin system (RAS) has been involved in sperm function, even so, little is known about the implication of one of the RAS axis formed by Ang-(1–7) (angiotensin-(1–7)) and MAS receptor. Hence, in the present work, we focused on elucidating the function of the MAS receptor in human spermatozoa. We analyzed the expression and localization of MAS receptor in human spermatozoa and we observed if its activation is able to modulate the sperm motility of normal motility and/or asthenozoospermic patients, as well as, the acrosome reaction of the spermatozoa. MAS receptor is present in human mature spermatozoa, not only at the mRNA level but also at protein level. MAS is localized at the acrosome region, as well as, in the tail of spermatozoa. The sperm incubation with MAS agonist Ang-(1–7) activates at dose-dependent manner the PI3K/AKT pathway (P P P

(Pro)renin receptor (PRR) is a protein that takes part in several signaling pathways such as Renin Angiotensin System and Wnt signalling. Its biological role has recently been related to cancer progression and in this study, we investigated its relevance in colorectal cancer (CRC). To that end, we analysed the immunohistochemical expression of PRR in adenomatous polyps and CRCs from the same patients (n = 42), and in primary tumours and nodal and liver metastases from advanced CRC patients (n = 294). In addition, the soluble fraction of PRR was measured by ELISA in plasma samples from 161 CRC patients. The results showed that PRR expression was gradually augmented along the uninvolved mucosa&ndash, adenoma&ndash, adenocarcinoma sequence. Besides, the stronger expression of PRR in primary tumours was markedly associated with local tumour extent and the onset of metastases. Moreover, PRR expression in both primary and distant metastases was associated with worse 5- and 10-year survival of CRC patients. Plasmatic PRR levels did not change with respect to controls and were not associated with CRC aggressiveness. These results suggest a key role of PRR in the development and progression of CRC and a potential use of this protein as a new prognostic biomarker and/or therapeutic target for this disease.

Background Aminopeptidase N (APN; EC 3.4.11.2) is a membrane dimeric metallopeptidase involved in differentiation, development, and proliferative processes of several tissues. Recent studies have demonstrated the increased expression and activity of this enzyme in several cancers. However, there are no available data about the impact of this peptidase in the biological aggressiveness and the survival of colorectal cancer (CRC) patients. Methods The activity and mRNA expression of APN in tumor tissue (n = 81) and plasma (n = 40) of patients with CRC of low and high grades and stages were prospectively analyzed by fluorimetric and quantitative reverse transcriptase–polymerase chain reaction methods. Data obtained in adenoma and CRC were compared with those from the surrounding normal mucosa. Classic clinical and pathological parameters were stratified following APN data and analyzed for 5-year survival. Results mRNA levels of APN ( ANPEP) were lower in colorectal adenomas and adenocarcinomas than in the surrounding uninvolved mucosa (Kruskal-Wallis, P < 0.001). Aminopeptidase N activity in CRC tissue was higher in patients with better overall survival (log-rank P < 0.05, Cox analysis P < 0.05). By contrast, higher plasmatic APN activity correlated with worse overall survival (log-rank P < 0.01, Cox analysis P < 0.05). Conclusions Aminopeptidase N activity in tissue and plasma from CRC patients is an independent prognostic factor of 5-year survival. The determination of APN activity levels in the plasma may be a safe, minimally invasive, and inexpensive way to define the aggressiveness of CRC in daily practice.

© 2015 Ivyspring International Publisher. Background and Objective: The role of peptidases in carcinogenic processes and their potential usefulness as tumor markers in colorectal cancer (CRC) have been classically attributed to cell-surface enzymes. The objective of the present study was to analyze the activity and mRNA expression of three cytosolic peptidases in the CRC and to correlate the obtained results with classic histopathological parameters for tumor prognosis and survival. Methods: The activity and mRNA levels of puromycin-sensitive aminopeptidase (PSA), ami-nopeptidase B (APB) and pyroglutamyl-peptidase I (PGI) were measured by fluorimetric and quantitative RT-PCR methods in colorectal mucosa and tumor tissues and plasma samples from CRC patients (n=81). Results: 1) PSA and APB activity was higher in adenomas and carcinomas than in the uninvolved mucosa. 2) mRNA levels of PSA and PGI was lower in tumors. 3) PGI activity in CRC tissue correlated negatively with histological grade, tumor size and 5-year overall suvival of CRC patients. 4) Higher plasmatic APB activity was independently associated with better 5-year overall survival. Conclusions: Data suggest that cytosolic peptidases may be involved in colorectal carcinogenesis and point to the determination of this enzymes as a valuable method in the determination of CRC prognosis.

The discovery of the intrarenal renin-angiotensin system (iRAS), which regulates angiogenesis, cell differentiation and proliferation, has opened new perspectives in the knowledge of kidney carcinogenesis. In this study we analyzed the immunohistochemical expression and fluorimetric activity of four key peptidases of iRAS in tumor tissue (n = 144) and serum samples (n = 128) from patients with renal neoplasms. Neutral endopeptidase (NEP/CD10), Angiotensin-converting enzyme-2 (ACE2), and aminopeptidase A (APA) were expressed in tumor cells whilst Angiotensin-converting enzyme (ACE) was expressed in the endothelial cells of intratumor blood vessels. The expression of ACE, ACE2 and NEP/CD10 was highest in clear cell renal cell carcinoma (CCRCC) and papillary renal cell carcinoma (PRCC). The expression of these enzymes correlated with CCRCC aggressiveness. In addition, NEP/CD10 correlated with 15-year overall survival. On the other hand, APA expression was decreased in CCRCC with higher grade and stage. The loss of expression of APA independently correlated with a worse 15-year overall survival. Serum activity of ACE2, NEP/CD10 and APA was significantly higher in renal tumor patients than in healthy subjects. Serum ACE activity was lower in high grade and metastatic CCRCC patients, and NEP/CD10 activity was negatively correlated with UISS (UCLA Integrated Staging System) and SSIGN (Mayo Clinic stage, size, grade and necrosis model) scores and with overall survival of CCRCC patients. These results suggest a metabolic imbalance of iRAS in renal tumors. This finding should be taken into account in the search of new diagnostic, prognostic and therapeutic tools for this disease. This work was partially funded by Grant SAF2013-48812-R from Ministerio de Economia y Competitividad (Spain), IT 8-11/13 from de Basque Government and UFI 11/44 from de University of the Basque Country (UPV/EHU). The current work has been developed as PhD project of PE and MB, who are recipients of a Predoctoral Fellowship from the Basque Government (Exp no PRE_2013_1_762 and PRE_2015_2_0148). This work was partially funded by Grant SAF2013-48812-R from Ministerio de Economia y Competitividad (Spain), IT 8-11/13 from de Basque Government and UFI 11/44 from de University of the Basque Country (UPV/EHU). The current work has been developed as PhD project of PE and MB, who are recipients of a Predoctoral Fellowship from the Basque Government (Exp no PRE_2013_1_762 and PRE_2015_2_0148)

F1Fo-ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H+ flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H+ within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F1 and FO domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo.

Clear cell renal cell carcinoma (CCRCC) is a heterogeneous and complex disease that frequently develops distant metastases. Fibroblast activation protein (FAP) is a serine peptidase the expression of which in cancer-associated fibroblasts has been associated with higher risk of metastases and poor survival. The objective of this study was to evaluate the role of FAP in metastatic CCRCC (mCCRCC). A series of 59 mCCRCC retrospectively collected was included in the study. Metastases developed either synchronous (n = 14) or metachronous to renal disease (n = 45). Tumor specimens were obtained from both primary lesion (n = 59) and metastases (n = 54) and FAP expression was immunohistochemically analyzed. FAP expression in fibroblasts from primary tumors correlated with FAP expression in the corresponding metastatic lesions. Also, primary and metastatic FAP expression was correlated with large tumor diameter (>7cm), high grade (G3/4), high stage (pT3/4), tumor necrosis and sarcomatoid transformation. The expression of FAP in primary tumors and in their metastases was associated both with synchronous metastases and also with metastases to the lymph nodes. FAP expression in the primary tumor was correlated with worse 10-year overall survival. Immunohistochemical detection of FAP in the stromal tumor fibroblasts could be a biomarker of early lymph node metastatic status and therefore could account for the poor prognosis of FAP positive CCRCC. This work was partially funded by Grant SAF2013-48812-R from Ministerio de Economia y Competitividad (Spain), IT 8-11/13 from de Basque Government and EHUA14/25 from de University of the Basque Country (UPV/EHU). The current work has been developed as PhD project of PE and MB, who are recipients of a Predoctoral Fellowship from the Basque Government (Exp no PRE_2013_1_762 and PRE_2015_2_0148). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Aspartyl aminopeptidase (ASP; EC 3.4.11.21) is a widely distributed and abundant cytosolic enzyme that regulates bioactive peptides such as angiotensin II. It has been demonstrated that the expression and activity of this enzyme is modified in tissue and serum of patients with several types of cancer. However, the involvement of ASP in the neoplastic development and survival of patients with colorectal cancer (CRC) has not been analyzed to date. The activity and messenger RNA expression of ASP in tumor tissue (n = 71) and plasma (n = 40) of patients with CRC was analyzed prospectively using fluorometric and quantitative real-time polymerase chain reaction methods. Data obtained from tumor tissue were compared with those from the surrounding normal mucosa. Classic pathologic parameters (grade, stage, nodal invasion, distant metastases and perineural, lymphatic, and vascular invasion) were stratified following ASP data and analyzed for 5-year survival. ASP was upregulated in CRC tissues, and greater activity correlated significantly with the absence of lymph node metastases and with better overall survival. Inversely, greater plasmatic ASP activity was associated with worse overall and disease-free survival. Data suggest that ASP is involved in colorectal neoplasia and point to this enzyme as a potential useful diagnostic tool in clinical practice.

Background.Papillary thyroid carcinoma (PTC), follicular thyroid adenoma (FTA), and thyroid nodular hyperplasia (TNH) are the most frequent diseases of the thyroid gland. Previous studies described the involvement of dipeptidyl-peptidase IV (DPPIV/CD26) in the development of thyroid neoplasia and proposed it as an additional tool in the diagnosis/prognosis of these diseases. However, very little is known about the involvement of other peptidases in neoplastic and hyperplastic processes of this gland.Methods.The catalytic activity of 10 peptidases in a series of 30 PTC, 10 FTA, and 14 TNH was measured fluorimetrically in tumour and nontumour adjacent tissues.Results.The activity of DPPIV/CD26 was markedly higher in PTC than in FTA, TNH, and nontumour tissues. Aspartyl aminopeptidase (AspAP), alanyl aminopeptidase (AlaAP), prolyl endopeptidase, pyroglutamyl peptidase I, and aminopeptidase B activities were significantly increased in thyroid neoplasms when compared to nontumour tissues. AspAP and AlaAP activities were also significantly higher in PTC than in FTA and TNH.Conclusions.These data suggest the involvement of DPPIV/CD26 and some cytosolic peptidases in the neoplastic development of PTC and FTA. Further studies will help to define the possible clinical usefulness of AlaAP and AspAP in the diagnosis/prognosis of thyroid neoplasms.