Hematopoietic Stem Cells and Hypertension
Angiotensin II Regulation of Proliferation, Differentiation,
and Engraftment of Hematopoietic Stem Cells
Seungbum Kim, Michael Zingler, Jeffrey K. Harrison, Edward W. Scott, Christopher R. Cogle,
Defang Luo, Mohan K. Raizada
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Abstract—Emerging evidence indicates that differentiation and mobilization of hematopoietic cell are critical in the
development and establishment of hypertension and hypertension-linked vascular pathophysiology. This, coupled
with the intimate involvement of the hyperactive renin–angiotensin system in hypertension, led us to investigate the
hypothesis that chronic angiotensin II (Ang II) infusion affects hematopoietic stem cell (HSC) regulation at the level
of the bone marrow. Ang II infusion resulted in increases in hematopoietic stem/progenitor cells (83%) and long-term
HSC (207%) in the bone marrow. Interestingly, increases of HSCs and long-term HSCs were more pronounced in the
spleen (228% and 1117%, respectively). Furthermore, we observed higher expression of C–C chemokine receptor type
2 in these HSCs, indicating there was increased myeloid differentiation in Ang II–infused mice. This was associated
with accumulation of C–C chemokine receptor type 2+ proinflammatory monocytes in the spleen. In contrast, decreased
engraftment efficiency of GFP+ HSC was observed after Ang II infusion. Time-lapse in vivo imaging and in vitro Ang
II pretreatment demonstrated that Ang II induces untimely proliferation and differentiation of the donor HSC resulting
in diminished HSC engraftment and bone marrow reconstitution. We conclude that (1) chronic Ang II infusion regulates
HSC proliferation, mediated by angiotensin receptor type 1a, (2) Ang II accelerates HSC to myeloid differentiation
resulting in accumulation of C–C chemokine receptor type 2+ HSCs and inflammatory monocytes in the spleen, and
(3) Ang II impairs homing and reconstitution potentials of the donor HSCs. These observations highlight the important
regulatory roles of Ang II on HSC proliferation, differentiation, and engraftment. (Hypertension. 2016;67:574-584.
DOI: 10.1161/HYPERTENSIONAHA.115.06474.) Online Data Supplement
•
Key Words: angiotensin II
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bone marrow transplantation
hypertension ■ inflammation
R
ecent evidence indicates that the renin–angiotensin system (RAS) plays critical roles in the development of the
hematopoietic system1–4 and hematopoiesis.5–7 Components of
the RAS, including angiotensinogen, angiotensin II (Ang II),
angiotensin 1 to 7 (Ang [1–7]), angiotensin-converting
enzyme, angiotensin-converting enzyme 2, angiotensin receptor type 1a (AT1R), AT2R are all present in bone marrow (BM)
cells.8,9 Several studies have shown that angiotensin-converting
enzyme inhibitors and AT1R/AT2R antagonists induce abnormal hematopoiesis suggesting that RAS regulates hematopoiesis through angiotensin receptors.5,10 In addition, Ang II and
Ang (1–7) have been demonstrated to influence proliferation
of hematopoietic progenitors and facilitate early recovery from
mild myelosuppression.11,12 Consistent with these are our previous studies demonstrating increases in BM proinflammatory
cells and a decrease in endothelial progenitor cells in chronic
Ang II–induced hypertension.13 These observations led us to
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hematopoietic stem cells
propose that Ang II would exert a profound influence in hematopoietic stem cell (HSC) at the BM level. Understanding
how Ang II regulates HSC would be critical as various BM
originated hematopoietic cells have been shown to contribute to initiation and progress of hypertension14–17 and hypertension-associated diseases, such as cardiac infarction and
arthrosclerosis.18,19
More than 1 million HSC transplantation (HSCT) were
performed around the world to correct a variety of BM deficiencies.20 During or after HSCT, immunosuppressive drugs
such as cyclosporine A are widely used to minimize the risk
of graft rejection and to increase the engraftment efficacy.21
However, their clinical use is frequently associated with 2to 5-fold increased Ang II level in the serum and kidney,
resulting in systemic and renal vasoconstriction that leads
to hypertension.22–24 Considering this side effect of immune
suppressors and prevalence of hypertension in public, it is
Received September 10, 2015; first decision October 4, 2015; revision accepted December 22, 2015.
From the Departments of Physiology and Functional Genomics (S.K., M.Z., M.K.R.), Pharmacology and Therapeutics (J.K.H., D.L.), Molecular
Genetics and Microbiology (E.W.S.), and Medicine (C.R.C.), College of Medicine, University of Florida, Gainesville.
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.
115.06474/-/DC1.
Correspondence to Mohan K. Raizada, Department of Physiology and Functional Genomics, College of Medicine, University of Florida, PO Box 100274,
Gainesville, FL 32610. E-mail [email protected]
© 2016 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.115.06474
574
Kim et al Regulation of Hematopoietic Stem Cell by Angiotensin II 575
critical to understand the role of RAS, especially the potent
effector Ang II on HSC homing and engraftment to enhance
HSCT efficiency. Engraftment of the donor-derived HSC
in lethally irradiated recipients involves dynamic and multistep processes.25 The donor HSCs recruited to the BM
go through transmarrow migration and lodge in the HSC
niche (HSC homing). Once the HSC arrives to the niche, its
expansion is orchestrated by a complex interplay of niche
cells, cytokines, and adhesion molecules in the microenvironment.26,27 Therefore, arrival of HSCs to the HSC
niche is critical for efficient reconstitution of hematopoiesis. Our second goal in this study was to know if Ang II
plays any important role in HSC homing and engraftment.
Taken together, it is of extreme importance to understand
how Ang II affects HSC regulation to enhance treatment of
hypertension and HSCT. Thus, our objectives in this study
were (1) to determine the effects of Ang II on proliferation
and differentiation of the most primitive HSC in vivo and
(2) to investigate whether Ang II affects HSC engraftment
efficiency.
Methods
Mice and Ang II Infusion
Male C57BL6 mice (2–3 months old) were purchased from
Charles River Laboratories. Human ubiquitin C promoter driven
GFP (UBC-GFP) and CX3CR1GFP/GFP mice were originally purchased from Jackson Laboratory and maintained at the University
of Florida. The latter mice were bred with C57BL6/J mice to generate CX3CR1+/GFP animals. Osmotic minipumps (1004, ALZAT
Corporation) were loaded either with Ang II (Bachem) dissolved in
0.9% saline (wt/vol) or with saline alone. Ang II was delivered at
a dose of 1000 ng·kg−1·min−1. Pumps were designed to administer
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Figure 1. Effect of chronic angiotensin II (Ang II) infusion on bone marrow (BM) hematopoietic stem/progenitor cells (HSPC) and long-term
hematopoietic stem cell (LT-HSC). A, Mean arterial pressure (MAP) measured by tail cuff after 3 weeks of Ang II infusion. B, The numbers
of average BM mononuclear cells (MNC) per 1 hind leg (1 femur and 1 tibia). C and D, Flow cytometric gating strategy for BM Sca-1+,
c-Kit+, Lin− (SKL; HSPC) and CD 150+, CD48− SKL cells (LT-HSC) in saline- and Ang II–infused mice. E, Percentile of HSPC in the BM.
F, The average of absolute numbers of total HSPC from 1 hind leg. G, Percentile of LT-HSC in the BM. H, The average of absolute
numbers of total LT-HSC from 1 hind leg; n=5 to 10 for each cohort. P values were designated as follows: *P≤0.05, **P≤0.01, ***P≤0.001.
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Figure 2. Effect of chronic angiotensin II (Ang II) infusion on spleen hematopoietic stem/progenitor cells (HSPC) and long-term
hematopoietic stem cell (LT-HSC). A, The average numbers of splenocytes from each spleen. B, Percentile of HSC in the spleen. C,
Absolute numbers of total HSC from each spleen. D, Percentile of LT-HSC in the spleen. E, The average of absolute number of total LTHSC from each spleen; n=5 to 10 for each cohort. *P≤0.05, **P≤0.01, ***P≤0.001.
Ang II or saline for at least 28 days, which were implanted subcutaneously into the dorsum. In some experiments, osmotic pumps
were replaced at the third week for constant saline/Ang II infusion.
Losartan (Sigma-Aldrich) was administered daily by intraperitoneal injection (20 mg·kg−1·day−1) All experimental procedures
performed on animals were in accordance with the University of
Florida’s Institutional Animal Care and Use Committee.
Video and Statistical Analysis
All videos were first captured using Volocity 5.5 and further processed
and edited with Apple iMovie and Sony Vegas Pro 9.0. Statistical significance was determined by Student’s t test using Prism 5 (Graphad). P values were designated as follows: *P≤0.05, **P≤0.01, and ***P≤0.001.
All values in the data are mean±SEM. All experimental protocols and
methods are available in the online-only Data Supplement.
Figure 3. Increased angiotensin receptor type 1a (AT1R) and C–C chemokine receptor type 2 (CCR2) expressions in hematopoietic stem/
progenitor cells (HSPC) and long-term hematopoietic stem cell (LT-HSC) of angiotensin II (Ang II)–infused mice. A, AT1R expressing cells
were analyzed in bone marrow (BM) Sca-1+, c-Kit+, Lin− (SKL) cells (left) and BM CD150+, CD48− SKL cells (right). B, The same AT1R
expressing cells were analyzed in spleen (SP). C, Fluorescence-activated cell sorting (FACS) contour plots showing CCR2+ cells (y axis) in
HSPC and LT-HSC of saline or Ang II–infused mice. *P≤0.05.
Kim et al Regulation of Hematopoietic Stem Cell by Angiotensin II 577
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Figure 4. Chronic angiotensin II (Ang II) infusion results in increased hematopoietic stem cell differentiation toward C–C chemokine
receptor type 2 (CCR2)+ myeloid/monocytic cells in the bone marrow (BM) and spleen. A, The average absolute numbers of CCR2+ and
CD11b+/Gr-1+/F4/80+ cells from each hind leg after 3-week saline/Ang II infusion (n=5–10). B, The average absolute numbers of CCR2+
and CD11b+/Gr-1+/F4/80+ cells from each spleen (SP). C, The average absolute numbers of CD11b+, Ly6Chi cells were further gated for
CCR2 expression from the BM cells of saline or Ang II–infused mice. D, The average absolute numbers of CX3CR1lo, Ly6Chi cells were
further gated for CCR2 expression from the BM cells. E and F, The same CCR2+,CD11b+, Ly6Chi cells and CCR2+,CX3CR1lo, Ly6Chi cells
were analyzed from splenocytes. *P≤0.05, **P≤0.01.
Results
Ang II Increases HSC in Both the BM and Spleen
Infusion of Ang II (1000 ng·kg−1·min−1) for 3 weeks in
C57BL6 mice resulted in 53 mm Hg increase in mean arterial pressure (102±8 mm Hg control versus 155±16 mm Hg
Ang II, Figure 1A). This effect was blunted by coadministration with losartan (20 mg·kg−1·d−1), an AT1R antagonist
(Figure 1A). The number of BM mononuclear cells in Ang
II–infused mice was increased by 16% (Figure 1B). In the BM
of normotensive mice, Sca-1+, c-Kit+, Lin− (SKL) cells that
are highly enriched for hematopoietic stem/progenitor cells
(HSPC) represented ≈0.24% of the BM mononuclear cells
(Figure 1C). The HSPC population was increased by 83% in
Ang II–treated mice (Figure 1D–1F). Coadministration with
Ang II and losartan significantly attenuated the increase of
SKL cells (Figure 1A, 1E, and 1F). We further purified the
long-term HSC (LT-HSC) from SKL cells by CD150+ and
CD48− selection. LT-HSC is a rare population of HSC in the
BM (≈0.002% of total BM cells, Figure 1C and 1D) that has
been shown to be normally quiescent but possesses life-long
hematopoietic repopulation potentials.28,29 We observed 207%
increase of LT-HSC in the BM, which was also attenuated
by cotreatment with losartan (Figure 1G and 1H). These data
indicate that chronic Ang II infusion resulted in an increase
in HSC proliferation.
Next, we examined the levels of HSCs in the spleen.
Interestingly, Ang II treatment resulted in 22% increase in total
splenocytes (Figure 2A), 228% increase in HSC (Figure 2B
and 2C), and 1117% increase in LT-HSC in the spleen of Ang
II mice (Figure 2D and 2E). These increases were attenuated
by cotreatment with losartan. This demonstrates a significant increase of extramedullary hematopoietic activity in the
spleen of Ang II–treated animals.
Ang II Regulates HSC Differentiation
We investigated if the expression levels of AT1R changed in
HSPC and LT-HSC in Ang II–treated animals. We observed
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significant increases of AT1R expressions on HSCs of both
BM and spleen (Figure 3A and 3B). Next, we measured the
C–C chemokine receptor type 2 (CCR2) levels to determine
if Ang II caused differentiation of HSCs into further differentiated progenitors. A recent study showed that the CCR2
level in HSC is a critical marker for the initiation of HSC to
myeloid differentiation and that CCR2+ HSCs are the intermediate HSC (HSC) with no LT reconstitution potentials.30 FACS
analysis showed that CCR2+ HSC populations are markedly
increased in both BM and spleen of Ang II–infused animals,
showing Ang II acts as an important initiator of HSC differentiation (Figure 3C).
Because the CCR2+ HSC is thought to be the most
upstream contributor of myelopoiesis,30 we next investigated if the increase of CCR2+ HSCs in Ang II mice was
also associated with increased myeloid cells that expressed
CCR2. We observed overall increases of CCR2+ expressing
myeloid (CD11b+/Gr-1+/F4/80+) cells (49%–80% in the BM
and 58%–142% in the spleen; Figure 4A and 4B). When more
defined Ly6Chi monocytes (CD11b+, Ly6Chi) and inflammatory monocytes (CX3CR1lo, Ly6Chi) were analyzed and further gated for CCR2, we found that the number of monocytes
and their CCR2 expressions were consistently increased in
Ang II–infused mice. This suggests that Ang II affected downstream myeloid differentiation after HSC proliferation and
that CCR2 expression was the key signal for BM to spleen
mobilization.31,32
C57BL6 mice were reconstituted with HSPC from
the BM of CX3CR1 +/GFP mice to determine the origin of
increased myeloid cells in the BM and spleen (Figure 5A).
We observed an increase of CX3CR1+/GFP in Ang II–infused
BM (Figure 5B). In addition, CX3CR1+/GFP myeloid colonies were observed only in the Ang II–infused BM (arrows
in the BM). Furthermore, there was significant accumulation of CX3CR1+/GFP cells in the spleen with marked increase
of round cells with typical monocyte morphology primarily located in the marginal zone of the spleen (arrows in the
spleen). The result suggests that accumulation of myeloid/
monocyte cells in the BM and spleen originated from the
BM HSCs.
Aberrant Engraftment of HSC in Ang II–Treated
Mice
We next determined if effects of Ang II on proliferation, differentiation, and mobilization of HSC adversely
affected engraftment of HSC into the BM in the HSCT setting. Homing and engraftment of HSC into the BM were
compared between the saline-/Ang II–infused groups that
are lethally irradiated by 2 different methods. At first, we
injected the minimal survival number of HSPC from UBCGFP mice (200 GFP+ SKL cells, determined from a separate pilot study) with 2×105 whole BM cells into saline or
Ang II (1000 ng·kg−1·min−1) infused and lethally irradiated
mice. Survival rates of the chimeric mice were monitored
>30 days for engraftment success. Only 58% of the Ang
II–infused hypertension mice survived, whereas all salineinfused control mice were rescued (Figure 6A). This was
associated with a noticeable reduction in engraftment of the
donor HSC-derived GFP+ cells (24%–50% compared with
control), which was more prominent at the early engraftment
stage of HSC (day 7) than the later (Figure 6B).
Second, we used a time-lapse imaging technique of the
mouse tibial bone to directly track the HSC engraftment
over time in saline- and Ang II–infused mice.33 In salineinfused control group, individual HSCs that developed
into colonies at the later time points were found mainly
near the endosteum (Movie S1 in the online-only Data
Supplement), where osteoblasts and other microenvironmental cells are enriched to support HSC.25,34,35 These GFP+
SKL cells started to engraft and actively expand in the
osteoblastic HSC niche within 48 hours of injection, which
is a critical hallmark of functional HSC (Figure 6D).26 In
contrast, GFP+ SKL cells in Ang II–treated animals did not
localize to the HSC niche and rarely formed proliferative
colonies, suggesting that most HSCs were further differentiated and lost the engraftment potential (Figure 6E; Movie
Figure 5. The increased myeloid cells are originated from the bone marrow (BM) hematopoietic stem cell. A, A diagram showing
hematopoietic stem/progenitor cells (HSPC) transplantation from CX3CR1+/GFP mice (n=5). B, Histology of the femur (top) and spleen
(bottom) showing increased CX3CR1-GFP+ cells (arrows) after 3 weeks of Ang II infusion (all bars=100 μm). *P≤0.05.
Kim et al Regulation of Hematopoietic Stem Cell by Angiotensin II 579
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Figure 6. Inefficient and abnormal engraftment of hematopoietic stem cell (HSC) in angiotensin II (Ang II)–treated mice. A, The survival
rate of saline or Ang II–infused, lethally irradiated mice rescued with 200 GFP+ Sca-1+, c-Kit+, Lin− (SKL) cells and 2×105 whole bone
marrow (WBM) cells (n=12). B, BM reconstitution from 5×103 GFP+ SKL cells in saline or Ang II–infused recipients at days 7 and 14 after
transplantation. C, A diagram showing the process of in vivo tibia imaging. D and E, Time-lapse in vivo imaging of HSC engraftment
(arrows) in saline or in Ang II–infused recipients. F, Direct visualization of spleen engraftment (unit=mm). G, Colony-forming unit-spleen
(CFU-s) count in saline and in Ang II–infused recipients. H, GFP+ cells in the spleen of saline or Ang II–infused recipients at days 7 and 14
after transplantation. *P≤0.05, **P≤0.01. HSPC indicates hematopoietic stem/progenitor cells.
S1 in the online-only Data Supplement). Because extramedullary hematopoiesis in the spleen commonly occurs in
lethally irradiated mice to facilitate hematopoietic recovery,6 we examined the spleen of these 2 groups to find out
that there was a statistically significant reduction in the
number of colony-forming unit-spleen in Ang II–infused
mice (Figure 6F and 6G). Furthermore, there was a marked
decreases of the GFP+ cells in the spleens of these mice
demonstrating that overall engraftment of Ang II–infused
mice was inefficient compared with saline-treated controls
(Figure 6H).
Proliferation and Differentiation of HSC Result in
Decreased Engraftment in Ang II–Infused Mice
To further investigate how HSCs engraft differently in these
2 groups, we first used a BrdU label retaining assay to track
slow cycling HSCs that maintained stemness in saline- or
Ang II–infused mice (Figure 7A).36 Although many BrdU
retaining HSCs were observed near the osteoblastic HSC
niche of the saline-treated animals, few BrdU retaining cells
were observed at the same area in Ang II–infused animals
(Figure 7B). In vivo imaging of donor HSCs 18 hours after
cell injection also indicated that there was early cell division
of HSC in Ang II–treated mice (Figure 7C and 7D; Movie
S2 in the online-only Data Supplement). Although most of
the transplanted HSCs in saline-infused mice remained as
single cells at 18 hours after injection, ≈1 of 4 cells in Ang
II–infused mice were observed as dividing cells (Figure 5E).
Donor-derived SKL cells analyzed 10 days after injection
indicated that there was a significant decrease of remaining HSCs in Ang II–infused animals (Figure 7F). Taken
together, the results suggest that Ang II has adverse effects
on HSC homing and engraftment because of untimely proliferation and early differentiation before reaching to the
HSC niche.
Ang II Directly Affects LT Reconstitution Potential
of HSC
To exclude the possibility that hemodynamic changes or
any other indirect in vivo effects from systemic Ang II infusion influenced the engraftment efficiency, we incubated
GFP+ Lin− BM cells with or without Ang II for 18 hours
in vitro, sorted and injected SKL cells (GFP+ HSPC) into
lethally irradiated animals (Figure 8A). The recipients were
all normotensive and the GFP+ HSPC were exposed to Ang
II only in vitro before injection. The early survival rate of
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Figure 7. Untimely proliferation and differentiation of hematopoietic stem cell (HSC) result in poor engraftment in angiotensin II (Ang
II)–infused mice. A, A diagram showing the BrdU retention assay. Hematopoietic stem/progenitor cells (HSPCs) from BrdU fed UBC-GFP
mice were transplanted into C57BL6 recipients. B, Immunohistochemistry of femur from saline- and Ang II–infused mice. BrdU retaining
GFP+ cells (slow cycling HSC) were mostly observed near the endosteum of saline-infused mice (arrows, bar, 100 μm). C and D, In vivo
imaging of homing GFP+ HSC (arrows) 18 hours after HSC injection. E, Percentile of dividing cells observed in the tibia bone marrow
(BM; bar, 200 μm). F, GFP+ Sca-1+, c-Kit+, Lin− (SKL) cells in whole BM at day 10. **P≤0.01, ***P≤0.001. DAPI indicates 4′,6-diamidino-2phenylindole.
recipients that received Ang II–exposed HSPC was much
lower than the control (40% versus 90%, Figure 8B). The
rate of hematopoiesis from Ang II–exposed HSPC was also
significantly lower, confirming the direct effect of Ang II on
HSC engraftment (Figure 8C). In addition, we observed poor
BM reconstitution and a significant decrease of SKL cells in
mice that received Ang II–exposed HSPC (Figure 8D). We
performed serial transplantation of SKL cells from the first
recipients to further test whether Ang II truly affected the LT
reconstitution potential of the HSC.29 Although the control
HSPC still maintained the strong reconstitution potential in
the secondary recipients, the Ang II–exposed HSPC failed
to restore hematopoiesis and GFP+ peripheral blood disappeared completely within 8 weeks in the secondary recipients (Figure 8D). Ki67 staining showed that more HSPCs
were in active phases of the cell cycle when exposed to Ang
II in vitro, confirming the previous result of untimely proliferation observed in Ang II–infused animals. The results
indicate that Ang II directly and negatively affects LT reconstitution potential of HSC.
Discussion
The most significant finding of this study is that Ang II has
profound influence on the compositions of HSC and myeloid
progenitors. Ang II directly induced HSPC/LT-HSC proliferation and CCR2+ monocytes accumulation in the BM and
spleen, while impairing homing and engraftment of HSC in
the HSCT setting. We propose that these actions contribute to
hypertensive effects of Ang II and suggest that controlling the
RAS activity should be considered before HSCT to enhance
engraftment efficacy of HSC. We observed that Ang II markedly increased numbers of both HSPC and quiescent LT-HSC,
an effect attenuated by losartan, suggesting that upregulated
AT1R transactivated various tyrosine and nontyrosine kinase
receptors to carry out its pleiotropic effects for cell proliferation.37 Our finding that Ang II has direct action on the BM
HSC is supported by other reports showing the presence of
AT1R in various BM cells and HSC.7,8,10,38 In addition, AT1Rmediated signaling is known to play critical roles for myeloid
differentiation,39,40 highlighting the importance of Ang II in
HSC regulation. Although direct Ang II actions are clearly
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Figure 8. Serial transplantation assay to confirm the direct effect of angiotensin II (Ang II) on long-term reconstitution potential of hematopoietic
stem cell. A, A diagram showing the experimental approach. Lineage marker negative bone marrow (BM) mononuclear cells (MNC) from UBCGFP mice (GFP+ Lin− BM MNC) were incubated in vitro with media or media+Ang II for 18 hours, sorted for hematopoietic stem/progenitor
cells (HSPC) and injected into lethally irradiated recipients with 106 BM MNC from C56BL6 mice. B, The survival rate of the primary recipients
that had either HSPC incubated with STEMSPAN media or HSPC incubated with media and Ang II (250 μg/mL) for 18 hours (n=10). C,
GFP+ peripheral blood (PB) reconstitution of each group on weeks 3 and 6. D, Serial transplantation assay using HSPC exposed to Ang II
before injection. Ang II–exposed HSPC showed significantly lower BM engraftment and did not reconstitute peripheral blood in the second
transplantation. E, Ki67+ HSPCs that are in active phases of the cell cycle after 18 hours in vitro culture with or without Ang II. *P≤0.05, **P≤0.01.
evident from our data, its indirect effects on other regulatory
microenvironmental cells in the BM such as osteoblasts41,42
and mesenchymal stem cells43,44 or extracellular matrix such
as collagen, which is an important structural component of the
HSC niche,45,46 cannot be ruled out at the present time.
Most of the HSCs reside within the BM, while few are
found in the spleen and circulation. These peripheral HSCs
can contribute to extramedullary hematopoiesis in pathological conditions, such as infection, cardiovascular diseases, or
irradiation.19,32,47–49 Although HSCs in the spleen resemble BM
HSCs because they are capable of multilineage reconstitution,47
LT-HSCs that have life-long hematopoiesis potentials are
thought to exist in the BM, based on observations that only the
BM has all microenvironmental cells that constitute the HSC
niche for LT-HSC maintenance.27,50 Interestingly, LT-HSCs that
were extremely rare in the spleen of control mice were readily
detectable in Ang II–infused mice, with an 8.6-fold increase in
percentile and an 11-fold increase in absolute number (Figure 2).
We also observed accumulation of cells with myeloid lineage
and inflammatory monocytes along with the increase of CCR2+
HSC. CCR2 is an important signal for recruiting hematopoietic
cells to the inflammatory sites31 and it could have played similar roles for mobilization of HSC to the spleen (Figures 3–5).32
In addition, we speculate that the oxidative stress from Ang II
infusion could have induced CCR2 overexpression and
myeloid-biased differentiation.51,52
Our study is unique in a way that it uses continuous
time-lapse in vivo imaging at a single-cell resolution, allowing direct observation of functional bona fide HSC with
Figure 9. Multiple effects of angiotensin II (Ang II) on hematopoietic
stem cell (HSC) regulation. Ang II initiated proliferation/
differentiation of bone marrow (BM) long-term HSC (LT-HSC)
leading to the increases and accumulation of C–C chemokine
receptor type 2 (CCR2)+ intermediate HSC (IM-HSC), hematopoietic
stem/progenitor cells (HSPC), and monocytes to the spleen. The
increased CCR2+ proinflammatory cells are proposed to facilitate
the progresses of vascular inflammation, neuroinflammation, and
hypertension-associated cardiovascular diseases. Ang II also
triggered premature proliferation of HSC that had adverse effects
on BM homing, resulting in decreased engraftment efficiency.
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engraftment and proliferation potentials (Figure 6). Using
this technology, we have shown that Ang II infusion significantly inhibited homing and engraftment of HSC into
the BM HSC niche. In vivo imaging showed that many
donor-derived HSC went through early proliferation possibly from the proliferative effect of Ang II through AT1Rmediated mean arterial pressure kinases and the JAK/STAT
activation.37 This untimely event resulted in a decrease of
available HSC for engraftment in the BM osteoblastic HSC
niche (Figure 7). As the microenvironmental signals that
HSC receive from the niche are critical for efficient engraftment and proliferation,27 this led to abnormal and inefficient
engraftment of HSC in Ang II–treated recipients. Because
in vitro exposure of Ang II to HSCs also negatively affected
engraftment in normotensive recipients, the results highlight
the direct and adverse role of Ang II for HSC engraftment
potentials (Figure 8).
We have summarized our conclusion in Figure 9, describing the important dual roles of Ang II in HSC regulation.
Although chronic Ang II infusion increased the numbers of
HSCs leading to myeloid-biased differentiation in the BM and
mobilization of CCR2+ HSCs/monocytes to the spleen, it also
decreased engraftment efficiency of HSC in the lethally irradiated recipients because of early differentiation and undue
proliferation before homing. It would be interesting to investigate the roles of increased myeloid progenitors and monocyte
in the progress of Ang II–induced hypertension. There is a
growing body of evidence that immune changes and increased
inflammatory monocytes are the key to development of hypertension and cardiovascular diseases through vascular inflammation.15,53 In addition, our previous evidence has shown that
neuroinflammation plays important roles in the development
and establishment of neurogenic hypertension.13,54 On the basis
of our present data, it is tempting to speculate that increases
of HSC and myeloid progenitors in the BM and spleen would
be critical to mobilization of these proinflammatory cells into
the brain.14,55,56
Perspectives
Our study shows that Ang II has profound influence on BM
and spleen HSCs, affecting their proliferation, differentiation, and engraftment efficiency. Considering that the level
of circulating Ang II can change drastically in patients with
hypertension,57 these changes may similarly affect human BM
and spleen, contributing to the progress of hypertension 16,17
and hypertension-associated cardiovascular diseases.18,19,30
Although we used the pressor dose to investigate immune
changes in the animal model, the effects of Ang II on hematopoietic cells may vary depending on the dose and exposure
time. Further study would be required to understand AT1R/
AT2R-mediated signalings that lead to immune changes in
hypertension. The negative effects of Ang II on HSC engraftment and homing have significant clinical implications in
HSCT, as Ang II–induced hypertension is one of the major
side effects of immunosuppressive drugs used after allogeneic HSCT.23 Angiotensin receptor blockers and angiotensinconverting enzyme inhibitors are commonly prescribed to
treat hypertension in patients with HSCT58 and our results
show that antihypertensive drugs should be carefully chosen
for these patients.59 There is no clinical data that has clearly
addressed the impact of hypertension on HSCT success and
patients survival. Thus, it would be relevant to undertake a
retrospective study to determine the influence of high blood
pressure in HSCT.
Acknowledgments
We gratefully acknowledge the help from Neal Benson and Dr
Vermali Rodriguez for data analysis.
Sources of Funding
This work was supported by National Institutes of Health grants
HL33610, DK105916, and HL56921.
Disclosures
None.
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Novelty and Significance
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What Is New?
Summary
• This study provides a direct evidence that angiotensin II (Ang II) regulates
Our study have shown that that Ang II is closely associated with
altered hematopoiesis and increased inflammation responses,
which required activation of different levels of hematopoietic stem/
progenitor cells. We showed that Ang II regulates primitive HSC
populations by increasing proliferation, myeloid-biased differentiation, and mobilization to the spleen through C–C chemokine
receptor type 2 expression. We used reconstitution assays and a
novel time-lapse in vivo imaging of the tibia to demonstrate that
Ang II impairs homing efficacy of HSC to the bone marrow stem cell
niche, resulting in poor hematopoietic reconstitution and survival
in lethally irradiated mice. The findings demonstrate the important
roles of Ang II in HSC regulation and may have clinical relevance in
hypertension treatment and HSC transplantation.
hematopoiesis in vivo at the stem cell level. Ang II infusion resulted in
increased number of C–C chemokine receptor type 2 expressing hematopoietic stem cell (HSC) and myeloid cells in the bone marrow and these
changes were even more significant in the spleen, suggesting that the
spleen can act as an important reservoir for both C–C chemokine receptor type 2+ HSC and inflammatory monocytes.
• Reconstitution assays and in vivo imaging of the tibia bone in lethally
irradiated mice showed that Ang II negatively affects the HSC homing to
its stem cell niche.
What Is Relevant?
• Ang II induced increases of C–C chemokine receptor type 2+ HSC and
myeloid progenitors in the bone marrow and spleen could contribute development of hypertension and cardiovascular diseases.
• As Ang II exposure triggers untimely proliferation and differentiation of
HSC resulting in poor engraftment, antihypertensive drugs that regulate
renin–angiotensin system should be carefully chosen for patients with
HSC transplantation.
Angiotensin II Regulation of Proliferation, Differentiation, and Engraftment of
Hematopoietic Stem Cells
Seungbum Kim, Michael Zingler, Jeffrey K. Harrison, Edward W. Scott, Christopher R. Cogle,
Defang Luo and Mohan K. Raizada
Downloaded from http://hyper.ahajournals.org/ by guest on June 17, 2017
Hypertension. 2016;67:574-584; originally published online January 18, 2016;
doi: 10.1161/HYPERTENSIONAHA.115.06474
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2016 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
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World Wide Web at:
http://hyper.ahajournals.org/content/67/3/574
Data Supplement (unedited) at:
http://hyper.ahajournals.org/content/suppl/2016/01/18/HYPERTENSIONAHA.115.06474.DC1
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Legend
Video 1 – HSC Engraftment in Saline Infused Mice
Video 2 – All mice had osmotic pumps filled with saline or Ang II for 1 week and then letally
irradiated 48 hours before HSC injection.
Online Supplement
Angiotensin II Regulation of Proliferation, Differentiation and Engraftment of
Hematopoietic Stem Cells
Seungbum Kim, 1 Michael Zingler, 1 Jeffrey K. Harrison,
Christopher R. Cogle, 4 Defang Luo, 2 and Mohan K. Raizada 1*
1
2
Edward W. Scott,
3
Department of Physiology and Functional Genomics, 2 Department of Pharmacology
and Therapeutics, 3 Department of Molecular Genetics and Microbiology, 4 Department
of Medicine, College of Medicine, University of Florida, Gainesville, FL
CORRESPONDENCE
Mohan K. Raizada, Ph.D., Distinguished Professor
Department of Physiology and Functional Genomics,
College of Medicine, University of Florida,
PO Box 100274, Gainesville, FL 32610
Phone: 352-392-9299
FAX: 352-294-0191
E-mail: [email protected]
Supplemental methods
FACS analysis and HSC sorting:
BM cells from UBC-GFP mice for transplantation were sex and age-matched. BM cells
were flushed from tibiae and femurs of both legs into PBS supplemented with 1% FBS,
2mM EDTA, 25mM HEPES (MACS buffer). Cells were centrifuged, passed through 20G
needle for single cell suspension and filtered through nylon mesh cell strainer. Cells
were treated with ACK buffer for 5-10 minutes on ice. The mouse lineage depletion kit
and AutoMACS (Miltenyi Biotec) were used as described in the manufacture’s protocol
to remove lineage positive cells after cell number determination. The lineage negative
cells were treated with FC block (Biolegend) for 10 minutes on ice. Combinations of
antibodies were used to further purify SKL or SLAM-SKL cells respectively (BD Science,
Biolegend and eBioscience). Antibodies used for FACS analysis were mouse Sca1(clone D7, PE-Cy7), c-Kit (clone 2B8, APC), CD150 (Clone TC15-12 F12.2, Pacific
Blue), CD48 (clone HM 48.1, PE), Ter119 (clone Ly-76, PE), Gr-1(clone RB6-8C5, PE),
B220 (clone RA3-6B2, PE), CD3 (clone 145 2C11, PE), CD4 (clone L3T4, PE), CD8
(clone 53-6.7, PE), CD11b (clone M1/70, PE) and Ly6C (clone HK1.4, Pacific blue). The
AT1R (Alexa Fluor 350) and CCR2 (PE)/CX3CR1 (APC) antibodies were from Bioss
and R&D systems respectively. Each population was sorted with BD FACS Aria II (BD
bioscience). For the BrdU retention assay, BrdU (800μg/ml) and sucrose (3% w/v) was
added to the drinking water of UBC-GFP mice for >2 month. GFP+ SKL cells from these
mice were injected into lethally irradiated mice infused with saline or Ang II. BrdU
retaining HSCs were stained with BrdU detection kit (Roche) according to the
manufacturers’ protocol.
Irradiation and HSC transplantation:
For the competitive repopulation assay, 1 week saline or Ang II infused C57BL6 mice
were given 950 rads of whole body irradiation (lethal dose). Subsequently, these mice
were transplanted with GFP+ SKL cells from UBC-GFP mice and C57BL6 whole BM
cells by retro orbital sinus (ROS) injection 48 hours following irradiation. Injected cell
numbers were carefully determined based on prescreening experiments of the mouse
survival and engraftment rates. For the serial transplantation assay, GFP + Lin- BM cells
from UBC-GFP mice were first cultured in vitro for 18h with STEMSPAN media
(Stemcell Technologies) with or without Ang II (250 µg/ml), sorted for GFP + SKL cells
and injected into lethally irradiated primary C57BL6 recipients (n=10). Four months later,
200 GFP+ SKL cells were sorted from the primary recipients from each group and
injected into the lethally irradiated secondary recipients (n=3-5). Enrofloxacin (Bayer)
were added to the drinking water during the first 2 weeks of engraftment to prevent
infection.
Tibia window installment:
One day before cell injection (D-1), the mice were anesthetized with 90 mg/kg of
Ketamine-HCl and 5mg/kg Xylazine-HCl. The hair around the surgical site was removed
and the mouse was positioned on the stage in the supine position and secured using
adhesive tape. After the mouse skin was disinfected with betadine, a small 5-7 mm
incision was made starting from the top of the tibia towards the ankle. After the tibia
bone was exposed, a sterilized drill bit attached to a rotary tool (Dremel) was used to
gently grind one side of the tibial surface to expose the marrow under a dissection
microscope (~5
magnification). The window was thinned sufficiently to allow
microscopic observation of the marrow.24 The average thickness of the ground bone
was ~40µm. This was done in only one leg.
In vivo imaging:
The first in vivo imaging was performed directly after tibia window installation while the
animals were still sedated to confirm transparency of the imaging area. Initial
engraftment observation was done within 12-24h after HSC injection. Mice were
anesthetized with avertin (250-400 mg/kg, Sigma-Aldrich) prior to in vivo imaging as it
increased the survival rate of the repeated in vivo imaging in lethally irradiated
recipients. Animals were placed on a disinfected and heated stage designed for the
microscope use. Images and videos were acquired with 5 or 10 magnification lens
(Apo, 0.15, 0.40 NA respectively) at room temperature using the DM5500B microscope
(Leica Microsystems), C7780 3 CCD camera (Hamamatsu) and Volocity 5.5 software
(PerkinElmer). To determine early proliferation, single or dividing cells from each cohort
(n=4) were analyzed 18h after HSC injection. After each imaging session, the open area
was closed with wound clips or surgical suture (Ethicon) and a plastic bandage was
applied around the leg to prevent damages.
Online supplementary video legend
Online Video S1. Time-lapse in vivo imaging of the mouse tibia bone showing different
HSC engraftment patterns in saline or Ang II infused mice. In saline infused
normotensive mice, GFP+ HSC tended to engraft near the endosteum of the bone.
These HSCs that arrived in the osteoblastic niche formed very tight and populous
colonies later (Day 4), which indicated their successful engraftment. In contrast, GFP +
HSCs in the Ang II infused mice rarely homed to their niches and showed very poor
reconstitution ability.
Online Video S2. Real time in vivo imaging of GFP+ HSC at 18h after cell injection.
Most HSCs existed as a single cell (54 of 58 observed cells, 93%) in the saline infused
mice (n=4), indicating that these cells were still migrating to the stem cell niches at this
stage. There were much fewer single HSCs (52 of 72 observed cells, 72%) at the same
time point in Ang II infused mice (n=4), showing that the HSCs went through early
proliferation before engraftment.