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Resumen del trabajo presentado en el 47º Congreso AEEH (Asociación Española para el Estudio del Hígado), celebrado en Madrid (España), del 9 al 11 de marzo de 2022, Background and Aims: Liver fibrosis is caused by an excessive accumulation of extracellular matrix (ECM) proteins. Macrophages are important effectors for ECM remodelling through phagocytosis and processing of the ECM within acidic compartments and can contribute to liver fibrosis resolution. Proteases, such as cathepsins, are essential for lysosomal proteolytic activity however, their contribution to ECM remodelling within the macrophages is unknown. Thus, the aim of this study was to investigate the proteolytic and degradative signalling pathways associated to macrophages during liver fibrosis. Method: To study the proteolytic and degradative signals contributing to fibrosis in macrophages we focused our attention on the lysosomal protease cathepsin D. We generated and validated a novel macrophage-CtsD knock-out mouse strain by breeding LysMCre (macrophages) with CtsD floxed mice. Fibrosis was established by bile duct ligation at 7 and 14 days in CtsDF/F or CtsDΔMac mice and determined by hydroxyproline, α-SMA, Col1A1 and TGF-β RT-PCR in total liver. Collagen degradation and endocytosis was studied using DQ™ Collagen, type I and 10KDa Dextran probes, respectively and WB for Endo180 and UPAR in peritoneal macrophages. Lysosomal colocalization was demonstrated using LAMP2. Protease secretome was determined using Protease Array from R&D. Results: First, CtsD deletion in macrophages from CtsDΔ Mac mouse was confirmed by CtsD WB and gene expression in macrophages and dual IHP (F4/80-CtsD) in liver tissue. Of note, cathepsin B expression remained unaffected despite deletion of CtsD. CtsD deletion in macrophages, increased liver fibrosis after BDL as shown by an increase in liver hydroxyproline, and hepatic α-SMA, Col1A1 and TGF-β gene expression. No differences of liver damage were detected between CtsDF/F and CtsDΔMac after BDL. As expected CtsD activity increased only in CtsDF/F mice after BDL. Isolated CtsDΔ Mac macrophages showed a significant decrease in DQ™ Collagen, type I degradation versus CtsDF/F ones. Collagen degradative profile colocalized partially with LAMP2, indicating that collagen was degraded within the lysosome. Furthermore, Dextran endocytosis and Endo180 and UPAR collagen internalization receptors remained unaffected in both CtsDF/F and CtsDΔ Mac macrophages. Protease array in cell culture medium from CtsDF/F and CtsDΔ Ma c macrophages revealed CtsA, MMP7, MMP8 and MMP13 as part of CtsD proteolytic secretome. Conclusion: Lysosomal cathepsin D is essential for a correct collagenolytic activity displayed by macrophages during liver fibrosis and its absence contributes to the development of cholestatic-induced liver fibrosis

Trabajo presentado en el International Workshop on Liver and Gut Fibrosis, celebrado en Valencia (España), los días 7 y 8 de octubre de 2021, Background and Aims: Changes in proteolytic activity are essential to liver fibrosis development. Previous reports suggest that macrophages are important effectors for ECM remodelling through phagocytosis and processing of ECM within acidic compartments. However, the role of macrophages in ECM remodelling during liver fibrosis is unknown. Methods: To study the degradative signals contributing to fibrosis in macrophages we focused our attention on the lysosomal protease cathepsin D (CtsD). We generated and validated a novel macrophage-CtsD knock-out mouse by breeding LysMCre (macrophages) with CtsD floxed mice. Fibrosis was established chronically by CCl4 (0.5μl/g) or bile duct ligation in CtsDF/F or CtsDΔMac mice and determined by SR staining, hydroxyproline and fibrogenic genes by RT-PCR. Collagen degradation and endocytosis was studied using DQ™ Collagen type I and 10KDa Dextran probes respectively in peritoneal macrophages. Lysosomal colocalization was determined by IF using LAMP2. WB for Endo180 and UPAR and dual IF (Collagen- F4/80) was performed in fibrotic livers. Results: First, CtsD deletion in macrophages from CtsDΔMac mouse was confirmed by WB and RT PCR in macrophages and dual IF (F4/80-CtsD) in liver tissue. Of note, cathepsin B expression remained unaffected despite deletion of CtsD. CtsDΔMac mice demonstrated enhanced liver fibrosis in both CCl4 and BDL models as shown by an increase in Sirius red staining, hydroxyproline and hepatic Col1A1 and TGF-β mRNA. CtsDΔMac macrophages showed a significant decrease in DQ™ Collagen type I degradation versus CtsDF/F ones. DQTM collagen profile partially colocalized within the lysosomes (LAMP2). No affectation of the endocytosis process in macrophages or the expression of the collagen receptors UPAR and Endo180 was detected in livers from CtsDF/F or CtsDΔMac after CCl4 challenge. Finally, F4/80+ Collagen loaded macrophages were detected in CCl4 livers. Conclusions: Lysosomal cathepsin D is essential for the correct collagenolytic activity displayed by macrophages during liver fibrosis.

Resumen del trabajo presentado en el International Liver Congress, celebrado de manera virtual del 23 al 26 de junio de 2021, Background and aims: Changes in proteolytic activity are essential to liver fibrosis development. Previous reports suggest that macrophages are important effectors for extracellular matrix (ECM) remodelling via its phagocytosis and processing within acidic compartments. However, the role of macrophages in ECM remodelling during liver fibrosis is unknown. Thus, the aim of this study was to investigate the proteolytic and degradative pathways associated to macrophages during liver fibrosis. Method: To study the degradative signals contributing to fibrosis in macrophages we focused our attention on the lysosomal protease cathepsin D (CtsD). We generated and validated a novel macrophageCtsD knock-out mouse by breeding LysMCre (macrophages) with CtsD floxed mice. Peritoneal macrophage polarization from CtsDΔMyel+/+ or CtsDΔMyel−/− mice was achieved with LPS for M1 or IL4/IL13 for M2 stimulation. Polarization markers and MMP profile were assessed by RT-PCR. Collagen degradation and endocytosis was studied using DQ™ Collagen, type I and 10 kDa Dextran probes respectively, and WB for Endo180 and UPAR. Lysosomal colocalization was determined using LAMP2. Fibrosis was established chronically by CCl4 (0.5μl/g) in CtsDΔMyel+/+ or CtsDΔMyel−/− mice and determined by SR staining, α-SMA IHP and Col1 WB as well as RT-PCR. Results: First, CtsD deletion in macrophages from CtsDΔMyel−/− mouse was confirmed by WB, CtsD activity in macrophages and dual IHP (F4/80-CtsD) in liver tissue. M1 and M2 polarization of CtsDΔMyel+/+ and CtsDΔMyel−/− macrophages using LPS (10 and 50 ng/ml) or IL4/ IL13 (20 and 50 ng/ml) resulted in similar and significant induction of iNOS (M1) and CD206 (M2) gene expression respectively. However, while M1 downstream effector CCl2 was similarly induced between CtsDΔMyel+/+ and CtsDΔMyel−/− macrophages after LPS stimulation, IL10 was defectively induced in M2-primed CtsDΔMyel−/− macrophages. In addition, both, M1 and M2 CtsDΔMyel−/− macrophages displayed defective induction of MMP-2, -3 and -7 gene expression. CtsDΔMyel−/− macrophages showed a significant decrease in DQ™ Collagen, type I degradation versus CtsDΔMyel+/+ macrophages at 37C. As expected, no degradation was detected at 4C. Collagen degradative profile colocalized partially with LAMP2, indicating that collagen was degraded within the lysosome. Furthermore, Dextran endocytosis in both CtsDΔMyel+/+ and CtsDΔMyel−/− macrophages remained unaffected. CtsDΔMyel−/− mice presented enhanced liver fibrosis as shown by an increase in Sirius red staining, alpha-SMA and Col1A1 gene and protein expression with no affectation of the Endo180/UPAR collagen internalization receptors (WB). Conclusion: Lysosomal cathepsin D is essential for a correct collagenolytic activity displayed by macrophages during liver fibrosis.

Trabajo presentado en las Jornadas CIBEREHD, celebradas online, del 9 al 11 de noviembre de 2020, Changes in proteolytic activity are essential to liver fibrosis development. Proteases control not only matrix turnover but also, the activation and repression of growth factors and chemokines influencing disease progression. Beyond metalloproteases, our knowledge of the proteolytic enzymes contributing to liver fibrosis is limited. The importance of lysosomal protease cathepsin D (CtsD) in kidney fibrosis has been recently demonstrated but its role in liver fibrosis remains elusive. Thus, the aim of this study was to analyse CtsD cells-specific role during liver fibrosis.

Acute kidney injury (AKI) is an abrupt reduction in kidney function caused by different pathological processes. It is associated with a significant morbidity and mortality in the acute phase and an increased risk of developing End Stage Renal Disease. Despite the progress in the management of the disease, mortality rates in the last five decades remain unchanged at around 50%. Therefore there is an urgent need to find new therapeutic strategies to treat AKI. Lysosomal proteases, particularly Cathepsin D (CtsD), play multiple roles in apoptosis however, their role in AKI is still unknown. Here we describe a novel role for CtsD in AKI. CtsD expression was upregulated in damaged tubular cells in nephrotoxic and ischemia reperfusion (IRI) induced AKI. CtsD inhibition using Pepstatin A led to an improvement in kidney function, a reduction in apoptosis and a decrease in tubular cell damage in kidneys with nephrotoxic or IRI induced AKI. Pepstatin A treatment slowed interstitial fibrosis progression following IRI induced AKI. Renal transplant biopsies with acute tubular necrosis demonstrated high levels of CtsD in damaged tubular cells. These results support a role for CtsD in apoptosis during AKI opening new avenues for the treatment of AKI by targeting lysosomal proteases.