X-linked hypophosphatemia (XLH/HYP)-with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscesses-is caused by mutations in the zinc-metallopeptidase PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization-inhibiting, acidic serine-and aspartate-rich motif (ASARM)-containing peptides, which are proteolytically derived from the mineral-binding matrix proteins of the SIBLING family (small, integrin-binding ligand N-linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motif-osteopontin (OPN) and bone sialoprotein (BSP)-as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full-length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an $35 kDa OPN fragment that was not present in wild-type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild-type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. In conclusion, these results identify full-length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization-inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP. ß

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Renin-angiotensin system (RAS) and kallikrein-kinin system (KKS) have a different site of interaction and modulate vascular tone and inflammatory response as well on exercise adaptation, which is modulated by exercise-induced cytokines. The aim of the study was to evaluate the role of ACE I/D and BDKRB2 +9/−9 polymorphism on exercise-induced cytokine response. Seventy-four male marathon finishers, aged 30 to 55 years, participated in this study. Plasma levels of exercise-induced cytokines were determined 24 h before, immediately after, and 24 h and 72 h after the São Paulo International Marathon. Plasma concentrations of MCP-1, IL-6 and FGF-21 increased after marathon in all genotypes of BDKRB2. IL-10, FSTL and BDNF increased significantly after marathon in the genotypes with the presence of the −9 allele. FSTL and BDNF concentrations were higher in the −9/−9 genotype compared to the +9/+9 genotype before (p = 0.006) and after the race (p = 0.023), respectively. Apelin, IL-15, musclin and myostatin concentrations were significantly reduced after the race only in the presence of −9 allele. Marathon increased plasma concentrations of MCP1, IL-6, BDNF and FGF-21 in all genotypes of ACE I/D polymorphism. Plasma concentrations of IL-8 and MIP-1alpha before the race (p = 0.015 and p = 0.031, respectively), of MIP-1alpha and IL-10 after the race (p = 0.033 and p = 0.047, respectively) and VEGF 72 h after the race (p = 0.018) were lower in II homozygotes compared to runners with the presence of D allele. One day after the race we also observed lower levels of MIP-1alpha in runners with II homozygotes compared to DD homozygotes (p = 0.026). Before the marathon race myostatin concentrations were higher in DD compared to II genotypes (p = 0.009). Myostatin, musclin, IL-15, IL-6 and apelin levels decreased after race in genotypes with the presence of D allele. After the race ACE activity was negatively correlated with MCP1 (r = −56, p < 0.016) and positively correlated with IL-8, IL-10 and MIP1-alpha (r = 0.72, p < 0.0007, r = 0.72, p < 0.0007, r = 0.47, p < 0.048, respectively). The runners with the −9/−9 genotype have greater response in exercise-induced cytokines related to muscle repair and cardioprotection indicating that BDKRB2 participate on exercise adaptations and runners with DD genotype have greater inflammatory response as well as ACE activity was positively correlated with inflammatory mediators. DD homozygotes also had higher myostatin levels which modulates protein homeostasis.

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Objective and design: Several proteases have drawn attention as potential targets to control the SARS-CoV-2 infection (COVID-19), thus circulating enzymatic activity and RAS regulation in severe hospitalized patients still remain to be determined. Material or subjects: 164 patients with COVID-19-like symptoms were grouped according to the severity of symptoms (COVID-19 negative, mild, moderate and severe). Methods: Patients were subjected to biochemical analyzes and to enzymatic activities of ACE2, ACE, DPPIV, PREP and CAT L, evaluated in serum samples. One-way ANOVA and multivariate logistic regression analysis were used. Statistical significance was accepted at p<0.05. Results: We show a correlation among comorbidities, elevated C-reactive protein (CRP) levels and disease severity. Additionally, concomitant high levels of D-dimer and CRP could be as prognostic for severe conditions. Assays of enzymatic activities revealed that, according to disease severity, both ACE2 and CAT L were statistically increased, while ACE, DPPIV and PREP activities were significantly reduced. Notably, analysis of ACE2/ACE ratio suggest a possible imbalance of Ang II/Ang1-7 ratio in severe patients. Conclusion: Our findings reveal the correlation between protease activity and the severity of COVID-19, in addition to highlighting the imbalance of ACE2/ACE ratio, predicting RAS dysregulation, closely related with a poor outcome of disease.

Background Anti-angiogenic drugs remain the mainstay therapy for several vascular retinal pathologies. The repurposing of approved anti-angiogenic drugs for use in ophthalmology can increase therapeutic options and reduce costs. The purpose of this study was to investigate the ocular safety profile of intravitreal (IVT) ramucirumab, an approved anti-vascular endothelial growth factor molecule for systemic treatment, using cell culture and animal models. Methods The cytotoxicity of ramucirumab at different concentrations was evaluated in human retinal pigment epithelial cells (ARPE-19) using the MTT assay. In addition, 250 or 500 µg of ramucirumab or vehicle was injected in the eye of 16 chinchilla rabbits. The eyes were evaluated by ophthalmoscopy, electroretinography, spectral-domain optical coherence tomography (SD-OCT) and by light and transmission electron microscopy. Results Electroretinography or SD-OCT did not detect functional or morphological alterations at 24 h or one week after injection. Light and transmission electron microscopy confirmed the absence of major signs of toxicity, although we found a statistically significant reduction in ganglion cell number between the controls and the eyes that received 500 µg of ramucirumab after 7 days. Compared to lower concentrations, 500 µg of ramucirumab caused reduction in cell viability and changes in morphology in ARPE-19 cells. Compared to the baseline, ocular and serum osmolarity showed no difference after IVT injection at all timepoints. Conclusion In conclusion, IVT injection of ramucirumab in rabbits is safe and does not cause functional damage to the retina. At the lower dose tested in vivo (250 µg), the morphology and ultrastructural anatomy were normal at 24 h and 1 week after the injection. However, the 500 µg dose can cause a decrease in ganglion cell number seven days after the injection.