Tendinopathies are common disabling conditions in equine and human athletes. The etiology is still unclear, although reactive oxygen species (ROS) and oxidative stress (OS) seem to play a crucial role. In addition, OS has been implicated in the failure of tendon lesion repair. Platelet-rich plasma (PRP) is rich in growth factors that promote tissue regeneration. This is a promising therapeutic approach in tendon injury. Moreover, growing evidence has been attributed to PRP antioxidant effects that can sustain tissue healing. In this study, the potential antioxidant effects of PRP in tenocytes exposed to oxidative stress were investigated. The results demonstrated that PRP reduces protein and lipid oxidative damage and protects tenocytes from OS-induced cell death. The results also showed that PRP was able to increase nuclear levels of redox-dependent transcription factor Nrf2 and to induce some antioxidant/phase II detoxifying enzymes (superoxide dismutase 2, catalase, heme oxygenase 1, NAD(P)H oxidoreductase quinone-1, glutamate cysteine ligase catalytic subunit and glutathione, S-transferase). Moreover, PRP also increased the enzymatic activity of catalase and glutathione S-transferase. In conclusion, this study suggests that PRP could activate various cellular signaling pathways, including the Nrf2 pathway, for the restoration of tenocyte homeostasis and to promote tendon regeneration and repair following tendon injuries.

Background. Equine adipose-derived mesenchymal stromal cells (e-AdMSC) exhibit attractive proregenerative properties strongly related to the delivery of extracellular vesicles (EVs) that enclose different kinds of molecules including RNAs. In this study, we investigated small RNA content of EVs produced by e-AdMSC with the aim of speculating on their possible biological role. Methods. EVs were obtained by ultracentrifugation of the conditioned medium of e-AdMSC of 4 subjects. Transmission electron microscopy and scanning electron microscopy were performed to assess their size and nanostructure. RNA was isolated, enriched for small RNAs (<200 nt), and sequenced by Illumina technology. After bioinformatic analysis with state-of-the-art pipelines for short sequences, mapped reads were used to describe EV RNA cargo, reporting classes, and abundances. Enrichment analyses were performed to infer involved pathways and functional categories. Results. Electron microscopy showed the presence of vesicles ranging in size from 30 to 300 nm and expressing typical markers. RNA analysis revealed that ribosomal RNA was the most abundant fraction, followed by small nucleolar RNAs (snoRNAs, 13.67%). Miscellaneous RNA (misc_RNA) reached 4.57% of the total where Y RNA, RNaseP, and vault RNA represented the main categories. miRNAs were sequenced at a lower level (3.51%) as well as protein-coding genes (1.33%). Pathway analyses on the protein-coding fraction revealed a significant enrichment for the “ribosome” pathway followed by “oxidative phosphorylation.” Gene Ontology analysis showed enrichment for terms like “extracellular exosome,” “organelle envelope,” “RNA binding,” and “small molecule metabolic process.” The miRNA target pathway analysis revealed the presence of “signaling pathways regulating pluripotency of stem cells” coherent with the source of the samples. Conclusion. We herein demonstrated that e-AdMSC release EVs enclosing different subsets of small RNAs that potentially regulate a number of biological processes. These findings shed light on the role of EVs in the context of MSC biology.

Equine penile squamous cell carcinoma (epSCC) is the most frequent tumor of the external male genitalia, representing 67.5% of equine genital cancers. epSCC is associated with papilloma virus (PV) infection and has been recently proposed as a model for human PV-induced squamous cell carcinomas. It has already been suggested that epSCC might undergo epithelial-to-mesenchymal transition (EMT). This work aims to investigate in detail this process and the possible role of PV oncoproteins in epSCC. For this purpose, 18 penile SCCs were retrospectively selected and tested for both EcPV2 presence and oncoproteins (EcPV2 E6 and EcPV2 E7) expression. Moreover, immunohistochemical EMT characterization was carried out by analyzing the main epithelial markers (E-cadherin, β-catenin, and pan-cytokeratin AE3/AE1), the main mesenchymal markers (N-cadherin and vimentin), and the main EMT-related transcription factors (TWIST-1, ZEB-1). PCR analysis was positive for EcPV2 in 16 out of 18 samples. EMT was investigated in epSCC positive for EcPV2. The immunohistochemistry results suggested the presence of EMT processes in the neoplastic cells at the tumor invasive front. Moreover, the significant upregulation of RANKL, together with BCATN1, LEF1, and FOSL1 genes, might suggest a canonical Wnt pathway activation, similarly to what is reported in human penile squamous cell carcinomas

Penile squamous cell carcinomas (SCCs) are common tumors in older horses, with poor prognosis mostly due to local invasion and recurrence. These tumors are thought to be mainly caused by Equus caballus papillomavirus type 2 (EcPV-2). The aim of this study is to characterize the tumor immune environment (TIME) in equine penile tumors. Equine penile epithelial tumors (17 epSCCs; 2 carcinomas in situ, CIS; 1 papilloma, P) were retrospectively selected; immune infiltrate was assessed by histology and immunohistochemistry; RT-qPCR tested the expression of selected chemokines and EcPV-2 DNA and RNA. The results confirmed EcPV-2-L1 DNA in 18/20 (90%) samples. L1 expression was instead retrieved in 13/20 cases (65%). The samples showed an increased infiltration of CD3⁺lymphocytes, macrophages (MAC387; IBA1), plasma cells (MUM1), and FoxP3⁺lymphocytes in the intra/peritumoral stroma when compared to extratumoral tissues (p < 0.05). Only MAC387⁺neutrophils were increased in EcPV-2^high viral load samples (p < 0.05). IL12/p35 was differentially expressed in EcPV^high and EcPV^low groups (p = 0.007). A significant decrease of IFNG and IL2 expression was highlighted in TGFB1-positive samples (p < 0.05). IBA1 and CD20 were intratumorally increased in cases where IL-10 was expressed (p < 0.005). EpSCCs may represent a good spontaneous model for the human counterpart. Further prospective studies are needed in order to confirm these preliminary results.

Physical exercise has been associated with the modulation of micro RNAs (miRNAs), actively released in body fluids and recognized as accurate biomarkers. The aim of this study was to measure serum miRNA profiles in 18 horses taking part in endurance competitions, which represents a good model to test metabolic responses to moderate intensity prolonged efforts. Serum levels of miRNAs of eight horses that were eliminated due to metabolic unbalance (Non Performer-NP) were compared to those of 10 horses that finished an endurance competition in excellent metabolic condition (Performer-P). Circulating miRNA (ci-miRNA) profiles in serum were analyzed through sequencing, and differential gene expression analysis was assessed comparing NP versus P groups. Target and pathway analysis revealed the up regulation of a set of miRNAs (of mir-211 mir-451, mir-106b, mir-15b, mir-101-1, mir-18a, mir-20a) involved in the modulation of myogenesis, cardiac and skeletal muscle remodeling, angiogenesis, ventricular contractility, and in the regulation of gene expression. Our preliminary data open new scenarios in the definition of metabolic adaptations to the establishment of efficient training programs and the validation of athletes’ elimination from competitions.