Showing papers by "Edward F. Srour published in 2018"

Tamm-Horsfall Protein Regulates Mononuclear Phagocytes in the Kidney

Abstract: Tamm-Horsfall protein (THP), also known as uromodulin, is a kidney-specific protein produced by cells of the thick ascending limb of the loop of Henle. Although predominantly secreted apically into the urine, where it becomes highly polymerized, THP is also released basolaterally, toward the interstitium and circulation, to inhibit tubular inflammatory signaling. Whether, through this latter route, THP can also regulate the function of renal interstitial mononuclear phagocytes (MPCs) remains unclear, however. Here, we show that THP is primarily in a monomeric form in human serum. Compared with wild-type mice, THP-/- mice had markedly fewer MPCs in the kidney. A nonpolymerizing, truncated form of THP stimulated the proliferation of human macrophage cells in culture and partially restored the number of kidney MPCs when administered to THP-/- mice. Furthermore, resident renal MPCs had impaired phagocytic activity in the absence of THP. After ischemia-reperfusion injury, THP-/- mice, compared with wild-type mice, exhibited aggravated injury and an impaired transition of renal macrophages toward an M2 healing phenotype. However, treatment of THP-/- mice with truncated THP after ischemia-reperfusion injury mitigated the worsening of AKI. Taken together, our data suggest that interstitial THP positively regulates mononuclear phagocyte number, plasticity, and phagocytic activity. In addition to the effect of THP on the epithelium and granulopoiesis, this new immunomodulatory role could explain the protection conferred by THP during AKI.

Megakaryocyte and Osteoblast Interactions Modulate Bone Mass and Hematopoiesis.

Abstract: Emerging evidence demonstrates that megakaryocytes (MK) play key roles in regulating skeletal homeostasis and hematopoiesis. To test if the loss of MK negatively impacts osteoblastogenesis and hematopoiesis, we generated conditional knockout mice where Mpl, the receptor for the main MK growth factor, thrombopoietin, was deleted specifically in MK (Mplf/f;PF4cre). Unexpectedly, at 12 weeks of age, these mice exhibited a 10-fold increase in platelets, a significant expansion of hematopoietic/mesenchymal precursors, and a remarkable 20-fold increase in femoral midshaft bone volume. We then investigated whether MK support hematopoietic stem cell (HSC) function through the interaction of MK with osteoblasts (OB). LSK cells (Lin-Sca1+CD117+, enriched HSC population) were co-cultured with OB+MK for 1 week (1wk OB+MK+LSK) or OB alone (1wk OB+LSK). A significant increase in colony-forming units was observed with cells from 1wk OB+MK cultures. Competitive repopulation studies demonstrated significantly higher engraftment in mice transplanted with cells from 1wk OB+MK+LSK cultures compared to 1wk OB+LSK or LSK cultured alone for 1 week. Furthermore, single-cell expression analysis of OB cultured±MK revealed adiponectin as the most significantly upregulated MK-induced gene, which is required for optimal long-term hematopoietic reconstitution. Understanding the interactions between MK, OB, and HSC can inform the development of novel treatments to enhance both HSC recovery following myelosuppressive injuries, as well as bone loss diseases, such as osteoporosis.

Absence of Cardiomyocyte Differentiation Following Transplantation of Adult Cardiac-Resident Sca-1+ Cells Into Infarcted Mouse Hearts.

Abstract: Although several lines of evidence suggest that the glycosyl phosphatidylinositol–anchored cell surface protein Sca-1 marks cardiac-resident stem cells, a critical analysis of the literature raises some concerns regarding their cardiomyogenic potential.1 Here, isolated adult cardiac-resident Sca-1+ cells were engrafted into infarcted hearts and monitored for cardiomyogenic differentiation. Donor cells were prepared from ACT–enhanced green fluorescent protein (EGFP); MHC-nLAC double-transgenic mice ([C57/Bl6J × DBA/2J]F1 genetic background; all procedures followed were in accordance with institutional guidelines). The ACTEGFP transgene targets ubiquitous expression of an EGFP reporter, and the MHCnLAC transgene targets cardiomyocyte-restricted expression of a nuclear-localized β-galactosidase reporter. Donor cell survival was monitored via EGFP fluorescence, whereas cardiomyogenic differentiation was monitored by reacting with the chromogenic β-galactosidase substrate 5-bromo-4-chloro-3-indolyl-β-d-galactoside (XGAL), which gives rise to a blue product.2 Double-transgenic hearts were dispersed with Blendzyme, and the resulting cells reacted with an allophycocyanin-conjugated anti–Sca-1 antibody and a phycoerythrin-conjugated cocktail of antibodies recognizing hematopoietic lineage markers.3 Sca-1+, EGFP+, lineage– cells were then isolated via fluorescence-activated cell sorting (characterization of the donor cells is provided in Figure [A]), and 100 000 cells were injected into the infarct border zone of nontransgenic [C57/Bl6J × DBA/2J]F1 mice immediately following permanent coronary artery occlusion. To screen for cardiomyogenic activity, hearts were harvested at 11 (n=3), 12 (n=3), 14 (n=2), or 21 (n=2) days postengraftment, fixed, and sectioned on a vibratome at 200 μm; sections were then reacted with X-GAL and visualized on a dissecting microscope. No nuclear β-galactosidase activity was detected (Figure [B]), despite the presence of Sca-1+ donor cells as evidenced by the presence of EGFP fluorescence (inset). To confirm the absence of cardiomyogenic differentiation, all vibratome sections exhibiting EGFP fluorescence were cryosectioned at 10 μm and analyzed (example shown in Figure [C]). No nuclear β-galactosidase activity was observed (8577 EGFP+ cells analyzed; the engraftment efficiency was 0.87%). Immune cytology confirmed that the green fluorescent signal colocalized with EGFP protein (not shown). As a positive control for the reporter system, cardiomyocytes prepared from embryonic day 15.5 ACT-EGFP; MHC-nLAC double-transgenic mice were engrafted into the hearts of syngeneic [C57/Bl6J × DBA/2J]F1 recipients; the hearts were harvested at 14 days postengraftment and processed exactly as described above. Donor cells with nuclear β-galactosidase activity and EGFP fluorescence were readily observed in the engrafted hearts (Figure [D]). Immune histology revealed that at least a portion of the Sca-1+ donor cells express vimentin (Figure Mark H. Soonpaa, PhD Pascal J. Lafontant, PhD Sean Reuter, BA John A. Scherschel, MD Edward F. Srour, PhD Marc-Michael Zaruba, MD Michael Rubart-von der Lohe, MD Loren J. Field, PhD

Megakaryocyte and Osteoblast Interactions Modulate Bone Mass and Hematopoiesis

Abstract: Emerging evidence demonstrates that megakaryocytes (MK) play key roles in regulating skeletal homeostasis and hematopoiesis. To test if the loss of MK negatively impacts osteoblastogenesis and hematopoiesis, we generated conditional knockout mice where Mpl, the receptor for the main MK growth factor, thrombopoietin, was deleted specifically in MK (Mplf/f;PF4cre). Unexpectedly, at 12 weeks of age, these mice exhibited a 10-fold increase in platelets, a significant expansion of hematopoietic/mesenchymal precursors, and a remarkable 20-fold increase in femoral midshaft bone volume. We then investigated whether MK support hematopoietic stem cell (HSC) function through the interaction of MK with osteoblasts (OB). LSK cells (Lin-Sca1+CD117+, enriched HSC population) were co-cultured with OB+MK for 1 week (1wk OB+MK+LSK) or OB alone (1wk OB+LSK). A significant increase in colony-forming units was observed with cells from 1wk OB+MK cultures. Competitive repopulation studies demonstrated significantly higher engraftment in mice transplanted with cells from 1wk OB+MK+LSK cultures compared to 1wk OB+LSK or LSK cultured alone for 1 week. Furthermore, single-cell expression analysis of OB cultured±MK revealed adiponectin as the most significantly upregulated MK-induced gene, which is required for optimal long-term hematopoietic reconstitution. Understanding the interactions between MK, OB, and HSC can inform the development of novel treatments to enhance both HSC recovery following myelosuppressive injuries, as well as bone loss diseases, such as osteoporosis.