ARTICLES Platelet-derived SDF-1 primes the pulmonary capillary vascular niche to drive lung alveolar regeneration Shahin Rafii1,2,3,4, Zhongwei Cao1,2,7, Raphael Lis1,2,4,7, Ilias I. Siempos2,5,7, Deebly Chavez1,2,3, Koji Shido1,2, Sina Y. Rabbany1,2,6 and Bi-Sen Ding1,3,8 The lung alveoli regenerate after surgical removal of the left lobe by pneumonectomy (PNX). How this alveolar regrowth/regeneration is initiated remains unknown. We found that platelets trigger lung regeneration by supplying stromal-cell-derived factor-1 (SDF-1, also known as CXCL12). After PNX, activated platelets stimulate SDF-1 receptors CXCR4 and CXCR7 on pulmonary capillary endothelial cells (PCECs) to deploy the angiocrine membrane-type metalloproteinase MMP14, stimulating alveolar epithelial cell (AEC) expansion and neo-alveolarization. In mice lacking platelets or platelet Sdf1, = PNX-induced alveologenesis was diminished. Reciprocally, infusion of Sdf1C C but not Sdf1-deficient platelets rescued lung regeneration in platelet-depleted mice. Endothelial-specific ablation of Cxcr4 and Cxcr7 in adult mice similarly impeded lung regeneration. Notably, mice with endothelial-specific Mmp14 deletion exhibited impaired expansion of AECs but not PCECs after PNX, which was not rescued by platelet infusion. Therefore, platelets prime PCECs to initiate lung regeneration, extending beyond their haemostatic contribution. Therapeutic targeting of this haemo-vascular niche could enable regenerative therapy for lung diseases. In mammals, surgical removal of the left lung by pneumonectomy cellular and molecular basis whereby MMP14 is specifically induced (PNX) leads to restoration of mass and gas exchange function in in the pulmonary vascular niche1,24,25 by PNX is not defined. the residual lung alveoli1–8. This process of compensatory lung Platelets are anuclear cells that constantly transit through the lung regrowth/regeneration results from formation of new functional blood vessels26,27. Activated platelets not only prevent haemorrhage alveolar units. PNX-induced neo-alveolarization is driven by by forming clots, but also promote organ repair through `inside-out' expansion of several cell types, which include alveolar epithelial cells mobilization of trophogens and adhesion molecules28–38. Enriched (AECs; refs 9–13), endothelial cells1,14–19 and mesenchymal cells3. with peptide and lipid mediators in granules39,40, platelets orchestrate However, it remains unknown how the loss of the lung lobe triggers multiple pathophysiological processes jointly with endothelial cells the alveolar regeneration in the remaining lungs. (ECs; refs 41,42). In response to stress, platelets are activated and Pulmonary capillary endothelial cells (PCECs) lining the lung adhere to the vascular bed of injured organs43–46. The intimate microvasculature form a specialized vascular niche to modulate the relationship between platelets and the lung vasculature26,33 set forth the function of alveolar progenitor cells11–18. Following PNX, upregulation hypothesis that resection of the left lung recruits circulating platelets of the membrane-type metalloprotease MMP14 (refs 20,21) in PCECs to prime the pulmonary vascular niche, driving alveolar regeneration. (ref. 1) leads to release of cryptic epidermal growth factor (EGF)- like ligands to stimulate the propagation of alveolar progenitor cells RESULTS such as type II alveolar epithelial cells (AEC2s; refs 9,10), initiating After PNX, recruited CD41C platelets are essential in initiating the regenerative alveolarization. Blockage of MMP14 activity by lung alveolar regeneration neutralizing antibody impeded the expansion of AECs but not PCECs, To test the pulmonary recruitment of platelets by PNX, we carried impairing neo-alveolarization and restoration of alveolar function. out flow cytometry analysis of platelet-specific marker CD41 (also However, the alveologenic function of endothelial-derived MMP14 known as ITGA2B) in total cells retrieved from the remaining lobes after PNX remains to be further demonstrated22,23. Moreover, the of pneumonectomized lungs. PNX caused significant enrichment of 1Ansary Stem Cell Institute, Weill Cornell Medical College, New York, New York 10065, USA. 2Department of Medicine, Weill Cornell Medical College, New York, New York 10065, USA. 3Department of Genetic Medicine, Weill Cornell Medical College, New York, New York 10065, USA. 4Department of Reproductive Medicine, Weill Cornell Medical College, New York, New York 10065, USA. 5First Department of Critical Care Medicine and Pulmonary Services, Evangelismos Hospital, University of Athens Medical School, Athens 10675, Greece. 6Bioengineering Program, Hofstra University, Hempstead, New York 11549, USA. 7These authors contributed equally to this work. 8Correspondence should be addressed to B-S.D. (e-mail: [email protected]) Received 7 April 2014; accepted 16 December 2014; published online 26 January 2015; DOI: 10.1038/ncb3096 NATURE CELL BIOLOGY VOLUME 17 | NUMBER 2 | FEBRUARY 2015 123 © 2015 Macmillan Publishers Limited. All rights reserved ARTICLES a b Retrieved total cells from the right lungs c PNX day 18 Left lung 104 Sham day 3 PNX day 3 Sham PNX Sham PNX l) 26.4% µ pneumonectomy Alveolar 24.5% 160 800 140 (PNX) regrowth 3 10 5.7% 120 600 100 2 80 400 P-selectin 10 6.1% 60 0 40 200 7.6% 20 P = 0.00028 P = 0.00017 2 3 4 2 3 4 0 Right lung volume ( 0 10 10 10 10 10 10 Right lung weight (mg) –/– –/– –/– –/– CD41 WT WT WT WT Thpo Thpo Thpo Thpo d e WT PNX day 18 PNX day 18 f Thpo–/– 1.0 Sham PNX 0.05 Sham PNX O) 0.8 2 0.04 0.6 0.03 0.4 P = 0.00055 0.2 0.02 (ml per cm H P = 0.00025 Lung compliance 0.01 –/– /– – – Inspiratory capacity (ml) – –/ –/ WT WT WT WT BrdU SFTPC VE-Cad DAPI Thpo Thpo Thpo Thpo PNX day 7 Retrieved lung total cells from g hipneumonectomized mice (PNX day 7) PNX day 7 5 –/– 10 105 WT Thpo PNX day 7 40 7.6% of 13.7% 3.4% cells 40 cells + 4 + 10 total cells 4 30 10 9.4% 30 3 10 3 1.7% population 20 10 population + 20 BrdU + SFTPC 102 10 102 10 0 27.3% 0 33.5% in SFTPC 102 103 104 105 –/– 0 in VE-Cad WT Thpo–/– 2 3 4 5 2 3 4 5 WT Thpo Percentage of Brdu 0 10 10 10 10 0 10 10 10 10 E-cadherin Percentage of Brdu VE-cadherin j k PNX PNX day 18 Cell propagation Platelet (PNX day 7) Aquaporin-5 Podoplanin DAPI depletion CD41 mAb (CD41 mAb injection Lung regrowth injection) every 3 days (PNX day 18) l m n PNX day 7 PNX day 7 Lung total cells (PNX day 7) cells cells 40 40 5 + Rat IgG CD41 mAb + 10 13.4% 30 30 104 2.1% 3.9% 9.2% population 3 + population 20 20 10 BrdU + 2 10 10 10 WT Thpo–/– 0 26.1% 31.2% 0 102 103 104 105 0102 103 104 105 in VE-Cad PNX day 18 in SFTPC Rat CD41 Rat CD41 o Percentage of BrdU Percentage of BrdU VE-cadherin IgG mAb IgG mAb Aquaporin-5 Podoplanin DAPI pqPNX day 18 PNX day 18 Sham PNX Sham PNX l) 800 160 µ 140 600 120 100 400 80 (mmHg) 2 60 200 P = 0.00016 40 paO 20 P = 0.0011 Right lung volume ( Rat CD41 Rat CD41 Rat CD41 Rat CD41 IgG mAb IgG mAb IgG mAb IgG mAb Rat IgG CD41 mAb Figure 1 After surgical removal of the left lung lobe by PNX, platelets are 40,6-diamidino-2-phenylindole. Characterization of the lungs of the sham essential for the regrowth/regeneration of the remaining right lung. (a,b) After mice is shown in Supplementary Fig. 1. (g,i) nD4 mice in all tested groups; PNX (a), surface expression of activation marker P-selectin on CD41C P D 0.00027 (g) and 0.0003 (i) between two groups. Scale bar, 50 µm. platelets was examined by flow cytometry (b). Sham-operated mice underwent (j) Distribution of AEC1s expressing aquaporin 5 and podoplanin in mice thoracotomy without lung resection. CD41CP-selectinC activated platelets are after PNX. (k–q) Lung regrowth in thromobocytopenic mice after PNX. Mice denoted in yellow (b). (c) Weight and volume of the right lungs in WT and TPO were injected with anti-CD41 mAb to deplete platelets (k). Propagation of null (Thpo−=−) mice lacking circulating platelets. Left panel: nD4 mice (both AEC2s (l) and PCECs (m,n), localization of AEC1s (o), right lung volume (p) sham groups), nD5 mice (WT with PNX) and nD4 mice (Thpo−=− with PNX). and arterial oxygen level (q) were measured in mice. (l,m) n D4 mice in all Right panel: nD5 mice (WT with sham), nD4 animals (Thpo−=− with sham) groups; P D0.00013 (l) and 0.00054 (m) between two groups. (p) nD3 mice and nD5 animals for both PNX groups. (d,e) Inspiratory capacity (d) and lung (IgG-treated sham), n D 5 mice (CD41 mAb-injected sham) and n D 5 mice compliance (e) in WT and Thpo−=− mice. nD4 mice (both sham groups), nD4 (both PNX); (q) nD4 mice (both sham), nD5 mice (PNX with IgG) and nD4 mice (WT with PNX) and n D 5 mice (pneumonectomized Thpo−=− group). mice (PNX with CD41 mAb). Scale bar, 50 µm. The error bar indicates s.e.m., (f–j) BrdU incorporation in SFTPCC AEC2s and VE-cadherinC PCECs was and the line represents the mean for c–e,g,i,l,m,p,q. The difference between tested in mice by immunostaining and flow cytometry, respectively. DAPI, individual groups was compared by unpaired two-tailed t-test. 124 NATURE CELL BIOLOGY VOLUME 17 | NUMBER 2 | FEBRUARY 2015 © 2015 Macmillan Publishers Limited. All rights reserved ARTICLES a PNX day 3 Sham day 3 b Total lung cells 105 PNX day 0 PNX day 3 PNX day 5 104 0.9% 27.7% 5.3% 3.4% 22.3% 103 1.3% SDF-1 102 0 3.1% 4.3% 7.6% 010102 103 4 105 010102 103 4 105 010102 103 4 105 SDF-1 CD41 VE-Cad DAPI CD41 cdeThpo–/– (PNX day 18) f Vehicle SDF-1 160 + –/– Thpo PNX 140 PNX 120 SDF1 35 + Sham 30 100 25 80 (mmHg) 20 2 15 60 SDF1 injection 10 paO 40 5 every 20 0 2 days platelets in the lungs 0 23