CD39 modulates hematopoietic stem cell recruitment and promotes liver regeneration in mice and humans after partial hepatectomy - PubMed (original) (raw)

. 2013 Apr;257(4):693-701.

doi: 10.1097/SLA.0b013e31826c3ec2.

Constanze Duhme 2, Wolfgang Junger 3, Steven D Salhanick 4 1, Yu Chen 3, Yan Wu 1, Vasilis Toxavidis 5, Eva Csizmadia 1, Lihui Han 1, Shu Bian 1, Günter Fürst 6, Martina Nowak 1 7, Seth J Karp 3, Wolfram T Knoefel 2, Jan Schulte Am Esch # 2, Simon C Robson # 1

Affiliations

CD39 modulates hematopoietic stem cell recruitment and promotes liver regeneration in mice and humans after partial hepatectomy

Moritz Schmelzle et al. Ann Surg. 2013 Apr.

Abstract

Objective: To study molecular mechanisms involved in hematopoietic stem cell (HSC) mobilization after liver resection and determine impacts on liver regeneration.

Background: Extracellular nucleotide-mediated cell signaling has been shown to boost liver regeneration. Ectonucleotidases of the CD39 family are expressed by bone marrow-derived cells, and purinergic mechanisms might also impact mobilization and functions of HSC after liver injury.

Methods: Partial hepatectomy was performed in C57BL/6 wild-type, Cd39 ectonucleotidase-null mice and in chimeric mice after transplantation of wild-type or Cd39-null bone marrow. Bone marrow-derived HSCs were purified by fluorescence-activated cell sorting and administered after hepatectomy. Chemotactic studies were performed to examine effects of purinergic receptor agonists and antagonists in vitro. Mobilization of human HSCs and expression of CD39 were examined and linked to the extent of resection and liver tests.

Results: Subsets of HSCs expressing Cd39 are preferentially mobilized after partial hepatectomy. Chemotactic responses of HSCs are increased by CD39-dependent adenosine triphosphate hydrolysis and adenosine signaling via A2A receptors in vitro. Mobilized Cd39 HSCs boost liver regeneration, potentially limiting interleukin 1β signaling. In clinical studies, mobilized human HSCs also express CD39 at high levels. Mobilization of HSCs correlates directly with the restoration of liver volume and function after partial hepatectomy.

Conclusions: We demonstrate CD39 to be a novel HSC marker that defines a functionally distinct stem cell subset in mice and humans. HSCs are mobilized after liver resection, limit inflammation, and boost regeneration in a CD39-dependent manner. These observations have implications for monitoring and indicate future therapeutic avenues.

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Figures

Figure 1

Figure 1

HSC proliferation and mobilization after partial hepatectomy in mice. A, HSC levels in the bone marrow of sham-operated wild-type and _Cd39_-null mice and 48 hours after 70% hepatectomy (% HSCs of bone marrow mononuclear cells; sham operation, n = 5; 70% hepatectomy, n = 8). B, HSC levels in the blood of sham-operated wild-type and Cd39_-null mice and 72 hours after 70% hepatectomy (% HSCs of peripheral blood mononuclear cells; sham operation, n = 5; hepatectomy, n = 5–9). C, HSC levels in the blood of sham-operated mice chimeric with wild-type or Cd39_-null bone marrow, as described earlier (% HSCs of peripheral blood mononuclear cells; sham operation, n = 2–3; 70% hepatectomy, n = 6–10). D, Cd39 expression by=HSCs in the blood of sham-perated wild-type mice and 72 hours after 70% hepatectomy (% Cd39-positive HSCs of total wild-type HSCs; n = 3). Error bars represent standard error of mean. *P < 0.05, †_P < 0.01, ‡_P < 0.001.

FIGURE 2

FIGURE 2

Bone marrow- and HSC-dependent liver regeneration. A, Hepatocyte proliferation in regular wild-type mice receiving 3 105 wild-type or _Cd39_-null HSCs 24 hours×after 70% hepatectomy (% Ki67-positive hepatocyte nuclei/high-power field relative to wild-type mice receiving phosphate-buffered saline as control; n = 6–8). B, Hepatocyte proliferation in bone marrow-irradiated mice chimeric with wild-type or _Cd39_-null bone marrow 72 hours after 70% hepatectomy (Ki67-positive hepatocyte nuclei/hpf; 70% hepatectomy, n = 5–10). C, Representative immunostaining for vascular P-selectin in livers of regular wild-type mice receiving 3 × 105 wild-type, _Cd39_-null HSCs or phosphate-buffered saline as control 24 hours after 70% hepatectomy (sham operation, n = 3; 70% hepatectomy, n = 6–8). Scale bars: 100 _μ_m. D, Representative immunostaining for vascular P-selectin in livers of bone marrow-irradiated mice chimeric with wild-type or Cd39_-null bone marrow 72 hours after 70% hepatectomy (n 6–10). Scale bars: 100 μ_m. E, interleukin-1_β_=plasma concentration (pg/mL) in bone marrow-irradiated mice chimeric with wild-type or Cd39_-null bone marrow at 72 hours (sham operation, n = 3; 70% hepatectomy, n = 6–8). F, interleukin-1_β concentrations (pg/mL) in liver sinusoidal endothelial cells, cultured for 5 hours with/without preincubation with adenosine triphosphate (5 mM) for 20 minutes. Error bars represent standard error of mean. *P < 0.05, †_P < 0.01, ‡_P < 0.001.

FIGURE 3

FIGURE 3

Chemotaxis of murine HSC. A, Migration rates of wild-type and _Cd39_-null HSCs (3 × 105 bone marrow mononu-clear cells) to vascular endothelial×growth factor (50 ng/mL) 4 hours after preincubation with adenosine triphosphate (100 _μ_M) or adenosine triphosphate (100 _μ_M) + soluble CD39 (solCD39) (5 U/mL; n = 5–6). B, Migration rates of wild-type and _Cd39_-null HSC toward vascular endothelial growth factor (50 ng/mL) after preincubation with ATP_γ_S (100 _μ_M; n = 6). C, Migration rates of wild-type and _Cd39_-null HSCs to vascular endothelial growth factor (50 ng/mL) after preincubation with adenosine (50 μ_M; n = 6). D, Migration rates of wild-type HSCs to vascular endothelial growth factor (50 ng/mL) after preincubation with adenosine triphosphate (50_μ_M) and selective adenosine receptor inhibitors [XAC-A1; CSC-A2A; alloxazine-A2B; or MRS1220-A3: (each 1.0 μ_M)] (n = 5–6). Error bars represent standard error of mean. †_P < 0.01, ‡_P < 0.001. 100% equals a mean cell number of n = ~ 1700.

Figure 4

Figure 4

Purinergic profiles of human CD133+ HSCs. A, mRNA expression of ectonucleotidases and ecto-nucleotide pyrophosphatases/ phosphodiesterases, purinergic P2X, P2Y, and P1 receptors by human HSCs relative to β_-actin (real-time reverse transciptase-polymerase chain reaction). B, Proportion of human blood HSCs (defined as % HSCs of peripheral blood mononuclear cells) 48 hours, 4 and 21 days after partial hepatectomy relative to levels before resection. Extent of liver resection was measured by computerized tomography-volumetry and defined as major liver resection (>30% of total liver volume; n_=6) and minor liver resection (<20% of total liver volume; _n_ = _18_). C, Correlation of HSC blood levels 24 hours after partial hepatectomy with the indocyanine green retention rate (15%) on postoperative day 21 (n = 13). D, Correlation of HSCs in the blood 24 hours after major hepatectomy (>30% of total liver volume) with reconstituted liver volume on postoperative day 21 (n = 6). E, CD39 expression by HSCs in the blood of patients after major liver resections (n = 5, >30% of total liver volume), gastrointestinal, that is, extrahepatic surgery (n = 5), or of healthy controls (n = 14). Data presented as CD39-positive to CD39-null ratio. Error bars represent standard error of mean. *P < 0.05, †_P < 0.01, ‡_P < 0.001.

FIGURE 5

FIGURE 5

Proposed mechanism of action. Chemotaxis of HSCs toward vascular endothelial growth factor is modulated by phosphohydrolysis of extracellular adeno-sine triphosphate by CD39 and stimulation of A2A receptor responses. CD39-positive HSCs are preferentially mobilized from the bone marrow to the liver. Decreases in adenosine triphosphate-driven paracrine interleukin-1_β_ signaling between liver sinusoidal endothelial cells and regenerating hepatocytes promote, at least in part, the observed increased hepatocyte turnover.

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