Session: 321. Coagulation and Fibrinolysis: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Bleeding and Clotting, Bleeding disorders, Hemophilia, Assays, Diseases, Biological Processes, Technology and Procedures, Multi-systemic interactions, Imaging
We developed, in collaboration with Jackson Laboratory, a new strain of hemophilic mice on the C57BL/6J background by knocking in a transgene encoding the fluorescent protein mScarlet under the endogenous F8 promoter. Using the CRISPR/Cas9 gene editing system, we inserted mScarlet into exon 1 of F8 through homology-directed repair. The animals showed a bleeding phenotype consistent with other HA mouse strains.
We first evaluated mScarlet expression in hepatocytes and liver ECs of mScarlet mice (n=3) using flow cytometry. After in situ perfusion via the portal vein, the livers were collected and processed into single-cell suspensions. An anti-CD31 antibody was used to identify ECs. Hepatocytes were identified by exclusion of CD45.2+ cells. CD31+ cells from mScarlet mice but not from control C57BL/6J mice expressed mScarlet. Neither mouse strain had detectable mScarlet in hepatocytes. We next evaluated mScarlet expression in LSECs located in different hepatic lobule zones. CD32bhiLSECs (which are located in zones 2 and 3, closest to the central capillary venule) showed the highest mScarlet expression, with ~30% of cells being mScarlet+. CD32b-/low LSECs (zone 1) showed markedly lower expression with only a small fraction of cells (~1%) being mScarlet+. We also detected mScarlet expression in non-LSEC hepatic ECs.
For fluorescence microscopy, we employed an anti-mScarlet antibody for improved detection. The hepatic staining pattern was consistent with that of the CD31 antibody, whereby the cells were in a sinusoidal arrangement of hepatic capillaries running between rows of hepatocytes toward central venules. We found mScarlet expression primarily in zones 2 and 3, with weaker signals in zone 1, consistent with the flow cytometry findings. Additionally, liver sections were co-stained for mScarlet and von Willebrand factor (vWF), the hepatic expression of which is restricted to LSECs. We found that mScarlet and vWF had the same staining pattern in the livers of the reporter mice. Liver sections from control mice showed vWF expression only.
We next used flow cytometry to investigate mScarlet expression in extrahepatic tissues. Liver lymphoid, mesenteric and inguinal lymph node, and lung, kidney, testicular, and splenic endothelial and lymphoid tissues of mScarlet were collected, processed, and analyzed. Kidney ECs, except for renal peritubular endothelial cells (RPECs), showed expression of mScarlet. However, kidney ECs showed ~5-fold lower mScarlet expression than liver ECs. The other tissues showed no mScarlet expression.
Kidney sections were then stained with anti-mScarlet and CD31 or vWF antibodies. We found that mScarlet was expressed in the kidneys of mScarlet mice in the same pattern as CD31 and vWF. No mScarlet expression was seen in the kidney sections of C57BL/6J HA mice lacking the reporter gene, which only showed anti-CD31 or anti-VWF antibody binding.
We propose that FVIII is primarily produced in LSECs located in zones 2 and 3 of hepatic lobules in the mouse. Additionally, FVIII is expressed in kidney ECs, but not in RPECs, hepatocytes, hepatic leukocytes, mesenteric or inguinal lymph nodes, or lung, kidney, testicular, and splenic ECs or leukocytes. However, expression in kidney ECs is considerably lower than in LSEC. Our novel mouse model expressing a well-defined and readily detectable reporter protein overcomes the challenges of immunohistochemistry using anti-FVIII antibodies, affording a better insight into the cellular origins of FVIII
Disclosures: Kaczmarek: Bayer: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BioMarin: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Spark: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Honoraria.
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