The Role of Rac Signaling in The Perivascular Niche

Transcription

1 The Role of Rac Signaling in The Perivascular Niche Felicia Ciuculescu Diaspora and Higher Education and Research Perspectives in Personalized Medicine- from Concept to Clinical Application Center for Gene and Cell Therapies of Cancer, Timisoara, Romania

2 Bone Marrow Stem Cell Niche Mesenchymal Stromal Cells: Nestin + MSCs:.8% of BM nucleated cells Immunopheno type: CD45 - CD31 - Ter119 - PDGFRα + CD51 + Murine: Lepr-Cre Cxcl12-GFP Nes-GFP, etc. Human: CD146 Immunopheno type: CXCL12 SCF Angp-1 VCAM-1 Nestin + cells required for HSCs/ progenitors homing Modified according to Morrison & Scadden. 214

3 Rac GTPases and their function in nonhematopoietic cells Rac GTPases: members of the Rho GTPase family and act as molecular switches integrating environmental signals. Mammalian Rac GTPases: Rac1, Rac2, Rac3 Rac1 is ubiquitously expressed Rac2 is expressed in hematopoietic cells Rac3 is widely expressed but mainly in CNS In vitro Inhibition of Rac1 promotes BMP2-induced osteoblastic differentiation (Oniashi et al. 213) Critical for OB adhesion, spreading, and proliferation (Jung et al. 211) Critical for MSCs differentiation into chondrocytes (Sosa-García et al. 21) Impaired OB migration (Fukuyama et al. 24) Rac1 function in osteoblasts (OB) WT In vivo OB-Rac del Trabecular microct Rac deletion from osteoblastic niche leads to reduced trabecular bone formation but normal hematopoiesis Lane et al. 211

4 Hypothesis The deletion of Rac in Nestin + cells will disrupt the perivascular hematopoietic niche and HSC function and hematopoiesis

5 Rac deletion in perivascular space affects: Vascular architecture Medullary bone formation Hematopoiesis

6 Isolation of Nestin + cells and confirmation of Rac1 excision in perivascular niche TG Rac1 / Rac3 -/- harvest BM isolate CD45- (MACS) stain CD45 CD31 Ter119 PDGFRα CD51 Rac1 fl/fl Rac1 / Rac1 wt TG WT 1 Nestin + cells 2 TG Rac1 / Rac3 -/- Rac1 fl/fl 3 4 water (-ve) PDGFRα + Nestin + cells CD51 + Nestin-Cre expression leads to partial deletion of Rac in vivo in perivascular bone marrow cells 7AAD + sort CD45 + CD31 + Ter119 +

7 Rac1 and Rac3 deficiency in perivascular niche leads to reduced number of Nestin + cells Nestin + cells in BM % of Nestin + cells (of BM).1.5. ** mean+/-sd, n=3,**p<.1

8 Rac1 and Rac3 excision in perivascular niche leads to reduced proliferative capacity and enhanced apoptosis of Nestin + cells PDGFRα+ Nestin + cells CD51+ # of cells (x1 4 ) % of appoptotic cells 2 TG WT TG Rac1 / Rac3 -/- 1 * day day 8 day 16 mean+/-sd, n=4,*p<.5 Annexin V 4 *** 2 mean+/-sd, n=3,***p<.1 *

9 Rac1 and Rac3 excision from perivascular niche leads to decrease number of Nestin + cells Nestin + cells participate in direct migration of HSC/progenitors (Mendez-Ferrer et al. 21)

10 Deficiency of Rac1 and Rac3 in the perivascular space is associated with reduced homing of LDBMs Methods C57BL6 mice DiD + homing effiency (%).2 isolate.1. LDBM stain Vybrant DiD Dye *** inject 2x1 6 LDBM cells/mouse 11.3 Gy 12hrs harvest Homed cells FACS Vybrant DiD BM Dye + cells TG WT TG Rac1 / Rac3 -/- mean+/-sd, n=15,***p<.1 Deletion of Rac genes in Nestin + cells in vivo impairs homing of HSC/progenitors to BM

11 Deficiency of Rac1 and Rac3 in the perivascular space leads to increase in sinusoidal and decrease in arteriolar distribution Laminin Endoglin Sca-1 Merged TG Rac1 / Rac3 -/- TG WT Laminin- arteries & sinusoids Sca-1- arteries Endoglin- sinusoids Shin-Young Park Kimberly Canty

12 Deficiency of Rac1 and Rac3 in the perivascular space leads to increased in sinusoidal volume and decreased in arteriolar volume % change in sinusoidal volume 2 * 1 % change in arteriolar volume 15 * 75 mean+/- SEM, n=4, p<.5 mean+/- SEM, n=4, p<.5

13 Rac1 and Rac3 deficiency in perivascular niche leads to increased and enlarged sinusoids PM Endoglin Laminin DAPI TG WT DIA DM 5µm 2µm 2µm PM Endoglin Laminin DAPI Frequency (%) 1 5 * ** ** TG Rac1 / Rac3 -/- DIA DM 5µm 2µm 2µm <1 µm 1-2 µm >2 µm sinusoid diameters (µm) mean+/-sem, n=3, *p<.5, **p<.1 Shin-Young Park Kimberly Canty

14 Rac1 and Rac3 deficiency in perivascular niche leads to increased vascular sinusoidal niche area contoured endoglin signal Endoglin DAPI TG WT TG Rac1 / Rac3 -/- 5µm 5µm 2µm 2µm 2µm 2µm 2µm 2µm % change in sinusoidal area 2 * 1 mean+/-sem, n=3, *p<.5 Shin-Young Park Kimberly Canty

15 Rac1 and Rac3 deficiency in perivascular niche leads to decrease in vascular arteriolar niche area TG WT TG Rac1 Rac3 -/- % change in arteriolar area 2 * 1 mean± SEM, n=3, *p<.5 Sca-1- arteries Shin-Young Park Kimberly Canty

16 Rac1 and Rac3 deficiency in perivascular space leads to increased trabecular bone formation femur TG WT coronal # of trabecule/ femur transverse sagittal 2 ** 1 TG Rac 1 / Rac3-/- % of trabecular bone volume TG WT TG Rac1 / Rac3-/- *** 6 3 TG WT TG Rac1 / Rac3-/-

17 Rac1 and Rac 3 deletion in Nestin + cells leads to increased in osteoblast progenitor cells 3 ** % of cells 15 * ALCAM Sca ALCAM+ Osteopontin (% relative to GAPDH) mean+/-sd, n=3,*p<.5, **p<.1 Sca-1+ The deletion of Rac1 and Rac3 in Nestin + cells upregulates osteopontin expression and increases osteoblast progenitors 3 15 **

18 Deficiency of Rac1 and Rac3 in the perivascular space is associated with decreased HSC and progenitor cell populations 2 Stem cells * BM Progenitors LSK (x1 4 ) LT-HSC (x1 3 ) ** BM- CFU/ 2.5x1 3 cells 2 ** 1 mean+/-sem, n=6,*p<.5, **p<.1 mean+/-sem, n=8,**p<.1, ***p<.1

19 Rac deletion in Nestin + cells decreases HSC and affects progenitors competitive repopulation capacity Competitors Rac del (TG Rac1 / Rac3 -/- ) CD45.1 CD45.1/2 CD45.2 Lethally irradiated recipients Donor derived cells in PB (%) 1 5 * * * wks 4 wks 8 wks 12 wks Competitor WT (CD45.1) TG Rac1 / Rac3 -/- (CD45.2) CD45.1 mean+/-sd, n=4, p<.1 CD45.2 Deletion of Rac genes in Nestin + cells impairs the donor-derived reconstitution capacity of HSPC

20 Deficiency of Rac1 and Rac3 in the perivascular space is associated with increased HSC and progenitor cell populations in spleen 1 ns 1 * LSK (x1 3 ) 5 LT-HSC (x1 3 ) 5 mean+/-sem, n=6,*p<.5, ns- not significant

21 Bone marrow perivascular stromal cells regulate HSCs maintenance in the hematopoietic microenvironment Nestin + cells, CAR cells and LepR + cells: perivascular stromal cells largely overlap (Mendelson and Frenette, 214) LepR + cells: main source of HSCs maintenance regulators, Scf and Cxcl12 (Zhou et al. 214) LepR + cells overlap with Nestin + GFP cells, contribute to bone and adipocyte regeneration (Mizoguchi et al. 214, Zhou et al. 214)

22 Rac1 and Rac3 excision in Nestin + cells leads to decrease in expression of HSC maintenance regulators, Scf and CXCL12 Normalized Scf mrna (%) Normalized m22 Scf mrna (%) 15 * * Normalized Cxcl12 mrna (%) * mean+/-sem, n=3,*p<.5

23 Conclusions Deficiency of Rac1 and Rac3 in the perivascular space is associated with: Decrease in Nestin + cells Decreased homing of WT hematopoietic cells into the medullary space Aberrant microvascular organization and medullary trabecular bone formation with reduced expression of factors supporting HSC Decrease in HSC and progenitor cell populations and their competitive repopulation capacity

24 Implication of the data Rac signaling is important for the interaction between HM constituents and HSCs and confirms the complexity of the HM with respect to the contribution of different cell types to the HSCs niche

25 Future aims to address experimental approaches Signaling pathways: AKT, JNK, STAT5, MAPK/PI3K Engraftment studies Nestin + cells: multi-potent capacity SNC interaction with hematopoietic microenvironment

26 Acknowledgments Williams Lab Prof Dr. David A Williams Serena DeVita Swaroopa Guda Pavan Nagaruri Gonchi Christian Brendel Meaghan McGuinness Chad Harris Silbestein Lab FACS Core Facility Scadden Lab Shin-Young Park Kimberly Canty Mahnaz Paktinat Ronald Mathieu David Scadden Vionne Yu Ebert s Lab Rebekka Schneider- Kramann

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