Platelet-derived SDF1 primes pulmonary capillary vascular niche to drive lung alveolar regeneration

Abstract

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 activated platelets trigger lung regeneration by supplying stromal cell-derived-factor1 (SDF1/CXCL12). After PNX, platelets stimulate SDF1-receptor CXCR4 and CXCR7 on pulmonary capillary endothelial cells (PCECs) to deploy membrane-type metalloproteinase MMP14, stimulating proliferation of alveolar epithelial cells (AECs) and neo-alveolarization. In mice lacking platelets or platelet Sdf1, PNX-induced alveologenesis was diminished. Reciprocally, infusion of Sdf1^+/+ 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 (Mmp14^iΔEC/iΔEC) exhibited impaired expansion of AECs but not PCECs, which could not be rescued by platelet infusion. Therefore, platelets prime PCECs to initiate lung regeneration, extending beyond their hemostatic contribution. Therapeutic targeting of this hemo-vascular niche could enable regenerative therapy for lung diseases.

Introduction

In mammals, surgical removal of the left lung by pneumonectomy (PNX) leads to restoration of mass and gas exchange function in the residual lung alveoli^{1-7, 70}. This process of compensatory lung regrowth/regeneration results from formation of new functional alveolar units. PNX-induced neo-alveolarization is driven by expansion several cell types that include alveolar epithelial cells (AECs)^8-12, endothelial^{1, 13-18}, and mesenchymal cells³. However, it remains unknown how the loss of lung lobe triggers the alveolar regeneration in the remaining lungs.

Pulmonary capillary endothelial cells (PCECs) lining the lung microvasculature form specialized vascular niche to modulate the function of alveolar progenitor cells^10-17. Following PNX, upregulation of membrane-type metalloprotease MMP14^{19, 20} in PCECs¹ leads to release of cryptic EGF-like ligands to stimulate the propagation of alveolar progenitor cells such as AEC2s^{8, 9}, initiating the regenerative alveolarization. Blockage of MMP14 activity by neutralizing antibody impeded the expansion of AECs but not PCECs, impairing neo-alveolarization and restoration of alveolar function. However, the alveologenic function of endothelial-derived MMP14 after PNX remains to be further demonstrated^{21, 22}. Moreover, the cellular and molecular basis whereby MMP14 is specifically induced in pulmonary vascular niche^{1, 23, 24} by PNX is not defined.

Platelets are anuclear cells that constantly transit through the lung blood vessel^{25, 26}. Activated platelets not only prevent hemorrhage by forming clots, but also promote organ repair via “inside-out” mobilization of trophogens and adhesion molecules^27-38. Enriched with peptide and lipid mediators in granules^{39, 40}, platelets orchestrate multiple pathophysiological processes jointly with endothelial cells (ECs)^{41, 42}. In response to stress, platelets are activated and adhere to the vascular bed of injured organs^43-46. The intimate relationship between platelets and the lung vasculature^{25, 33} set forth the hypothesis that resection of left lung recruits circulating platelets to prime vascular niche, driving alveolar regeneration.

Results

After PNX, recruited CD41⁺ platelets are essential in initiating lung alveolar regeneration

To test the pulmonary recruitment of platelets by PNX, we performed flow cytometry analysis of platelet-specific marker CD41 in total cells retrieved from the remaining lobes of pneumonectomized lungs. PNX caused significant enrichment of CD41⁺ platelets in the lungs after PNX (Fig. 1a, b). In control mice whereby thoracotomy was performed without lung resection (sham), there was negligible deposition of platelets. Surface expression of P-selectin, platelet activation marker, was detected in the majority of CD41⁺ platelets in the pneumonectomized lungs (Fig. 1b). Thus, activated platelets are recruited to the residual right lungs after surgical removal of left lung lobe.

Figure 1. After left lung pneumonectomy (PNX), platelets are essential for the regrowth/regeneration of the remaining right lungs.

a, b) After surgical removal of left lung lobe by PNX (a), surface expression of activation marker P-selectin on CD41⁺ platelets was examined by flow cytometry (b). Sham-operated mice underwent thoracotomy without lung resection. CD41⁺P-selectin⁺ activated platelets are denoted in yellow (b).

c) Weight (left) and volume (right) of the right lungs in wild type (WT) and thrombopoietin null (Thpo^−/−) mice lacking circulating platelets. Left panel: n = 4 mice (both sham groups), n = 5 mice (WT group underwent PNX), and n = 4 mice (Thpo^−/− group with PNX). Right panel: n = 5 mice (WT with sham), n = 4 animals (Thpo^−/− with sham), and n = 5 animals for both PNX groups.

d, e) Lung inspiratory volume (d) and compliance (e) were restored in WT but not Thpo^−/− mice at day 18 after PNX. n = 4 mice (both sham groups), n = 4 mice (WT with PNX), and n = 5 mice (pneumonectomized Thpo^−/− group).

f-j) Bromodeoxyuridine (BrdU) incorporation in SFTPC+ type 2 alveolar epithelial cells (AEC2s) and VE-cadherin⁺ pulmonary capillary endothelial cells (PCECs) was tested in mice by immunostaining and flow cytometry, respectively. Characterization of lungs of the sham mice is shown in suppl. Fig. 1. Panels (g) and (i): n = 4 mice in all tested groups; P = 0.00027 (g) and 0.0003 (i) between two groups. Scale bar = 50 μm. SFTPC, pro-surfactant protein C.

j) Distribution of type 1 alveolar epithelial cells (AEC1s) expressing aquaporin-5 and podoplanin in mice after PNX.

k-q) Lung regrowth and cell proliferation in thromobocytopenic mice after PNX. Mice were injected with anti-CD41 monoclonal antibody to deplete platelets (k). Propagation of AEC2s (l) and PCEC (m-n), localization of AEC1s (o), right lung volume (p) and arterial oxygen level (q) were measured in mice. Panels (l), (m): n = 4 mice in all groups; P = 0.00013 (l) and 0.00054 (m). Panel (p): n = 3 mice (IgG-treated sham), n = 5 mice (CD41 mAb-injected sham), and n = 5 mice (both PNX); Panel (q): n = 4 mice (both sham), n = 5 (PNX with IgG), and n = 4 (PNX with CD41 mAb). Scale bar = 50 μm. Error bar indicates stand error of mean (s.e.m.), and line represents mean for panels c, d, e, g, i, l, m, p, q. Difference between individual groups was compared by unpaired two-tail t-test between individual groups.

To assess the contribution of platelets to alveolar regrowth, we used Thrombopoietin-deficient (Thpo^−/−) mice in which the circulating platelet number is 5% of wild type (WT) littermate. In WT mice, PNX enhanced the mass, volume and respiratory function of the residual right lungs to a comparable level to the unresected sham lungs after 18 days (Fig. 1c-e). Notably, there was minor expansion of activated lung fibroblast cells in the residual right lungs after PNX, implicating the absence of fibrosis (suppl. Fig. 1). By contrast, this functional regrowth of the lungs was markedly reduced in Thpo^−/− mice (Fig. 1c-e). PNX-induced alveolarization is driven by the propagation of pro-surfactant protein C (SFTPC)⁺ AEC2s^{8, 9} and VE-cadherin⁺ PCECs that peaks at day 7 post PNX¹. Using BrdU incorporation, we found that the proliferation of AEC2s and PCECs induced by PNX were significantly lower in Thpo^−/− mice than that of WT mice (Fig. 1f-i, suppl. Fig. 1-2a). Reduced AEC2 proliferation in Thpo^−/− mice was associated with lower restoration of aquaporin-5⁺podoplanin⁺ type I AECs (AEC1s) in the pneumonectomized lungs (Fig. 1j), underscoring the potential of platelets to promote lung alveolar regeneration.

We then injected anti-CD41 monoclonal antibody (mAb) into WT mice to deplete platelets and subjected these thromobocytopenic mice to PNX (Fig. 1k, suppl. Fig. 2). Rat IgG treated mice were used as control. After PNX, recovery of volume, expansion of AECs and PCECs, and blood oxygenation were all inhibited in the remaining lungs of CD41 mAb-treated mice after PNX (Fig. 1l-q). Therefore, circulating platelets are essential for restoring the cellular components and respiratory function in the lungs after PNX.

Platelets deposit stromal cell-derived factor 1 (SDF1/CXCL12) to mediate PNX-induced neo-alveolarization

Activated platelet cells deploy chemokines such as SDF1/CXCL12 to promote organ repair^{23, 47-49}. Indeed, we found that the majority of CD41⁺ platelets associated with PCECs were stained as SDF1⁺ in the lung cryosection after PNX but not sham operation (Fig. 2a, suppl. Fig. 2e). This unexpected deposition of SDF1⁺CD41⁺ platelets on VE-cadherin⁺ PCECs reached the highest level at day 3 and sustained for at least 9 days after PNX (Fig. 2b-c). SDF1 belongs to CXC chemokine family^{34, 40, 50, 51} and plays a key role in modulating organogenesis^{47, 48, 52, 53}. Indeed, SDF1 injection significantly enhanced the recovery of AECs and restoration of gas exchange function in pneumonectomized Thpo^−/− mice (Fig. 2d-f). To examine the role of SDF1 in PNX-induced lung regrowth, we conditionally deleted the Sdf1 gene by crossing Sdf1^LoxP/LoxP mice⁵³ with tamoxifen-responsive ROSA-Cre^ERT2 mice. Treatment of the offsprings with tamoxifen led to inducible deletion of Sdf1 in adult mice (Sdf1^Δ/Δ) (Fig. 2g-i). Compared with control mice, Sdf1^Δ/Δ mice exhibited impaired AEC2 propagation and mitigated restoration of lung weight and volume after PNX (Fig. 2j, k). Thus, SDF1 signaling is critical for lung alveolar regeneration induced by PNX.

Figure 2. Pulmonary accumulation of platelets carrying stromal cell-derived factor 1 (SDF1/CXCL12) after PNX.

a) Immunostaining of SDF1, VE-cadherin, and platelet marker in mouse lung cryosections. CD41⁺SDF-1⁺ platelets were associated with VE-cadherin⁺ PCECs after PNX (inset). Scale bar = 50 μm.

b, c) Flow cytometry analysis of SDF1⁺ platelet recruitment in the remaining lungs after PNX. Red color depicts SDF1⁺CD41⁺ platelets, and small amounts of CD41⁻SDF1⁺ cells are presented in yellow color. Kinetics of platelet accumulation in the lungs after PNX is assessed (c). n = 6 animals in all time points.

d-f) Mouse alveolar regrowth in Thpo^−/− mice after injection of recombinant SDF1. Thpo^−/− mice were treated with SDF1 injection, and AEC1 distribution (e) and gas exchange function (f) treated mice was assessed by comparing with vehicle-injected group. n =4 mice in both groups; P = 0.0055 between two groups; Scale bar = 50 μm.

g-i) Inducible deletion of Sdf1 in adult mice (Sdf1^Δ/Δ). Mice expressing tamoxifen-responsive Rosa-Cre^ERT2 were crossed with Sdf1^loxP/loxP mice to generate Sdf1^Δ/Δ and control Sdf1^Δ/+ mice. SDF1 protein was determined by immunoblot in isolated platelets from mice 30 days after last injection. Protein level in indicated groups was compared after normalization to β-actin; Representative image is shown (i). SDF1 protein in Sdf1^Δ/Δ platelets is 3% of that of wild type mice (n = 6 mice in all genotypes).

j-k) Proliferation of AEC2s (j) and lung regrowth (k) in Sdf1^Δ/Δ and control mice after PNX. (k): n = 4 and 3 in sham-operated control and Sdf1^Δ/Δ mice, n = 5 and 4 in control and Sdf1^Δ/Δ mice group that underwent PNX. Scale bar = 50 μm. Error bar depicts s.e.m., and line stands for mean for panels c, f, h, k. Statistical difference between groups was assessed by unpaired two tail t-test.

We then formally deciphered the functional contribution of SDF1 from various niche cell components to lung regeneration (suppl. Fig. 3). The in vivo role of platelet-derived SDF1 was examined by breeding Platelet factor 4-Cre mouse line with floxed Sdf1 mice (Fig. 3a). This strategy specifically deletes Sdf1 in mouse platelets and platelet progenitors (Sdf1^ΔPLT/ΔPLT). Compared to control Sdf1^ΔPLT/+ mice, the restoration of lung mass and volume and regeneration of AECs and PCECs were significantly reduced in Sdf1^ΔPLT/ΔPLT mice (Fig. 3b-j). By contrast, deletion of Sdf1 in both myeloid and endothelial cells caused insignificant reduction in tested lung regenerative responses (suppl. Fig. 3). Notably, Sdf1^ΔPLT/ΔPLT mice did not exhibit altered production of growth factors from platelets, recruitment of hematopoietic cells, or pulmonary fibrin deposition after lung injury (suppl. Fig. 4). Hence, deployment of SDF1 from activated platelets is indispensible to elicit regenerative alveolarization after PNX (Fig. 3k).

Figure 3. Platelets recruited by PNX derive SDF1 to elicit alveolar re-growth/regeneration.

a-c) Platelet (PLT)-specific deletion of Sdf1 (Sdf1^ΔPLT/ΔPLT) in mice. Mouse line with platelet/platelet progenitor-specific Platelet factor 4 promoter-driven Cre (PF4-Cre) was crossed with Sdf1^loxP/loxP mice to delete Sdf1 specifically in platelets (a). Sdf1^ΔPLT/+ mice harboring haplodeficiency of Sdf1 in platelets served as control. SDF1 protein in platelets was analyzed by Western blot to determine Sdf1 deletion efficiency (b-c). SDF1 protein was decreased by 95% in Sdf1^ΔPLT/ΔPLT platelets, compared to wild type control (n = 6 mice per group).

d-e) Right lung weight (d) and volume (e) in Sdf1^ΔPLT/ΔPLT and control mice after PNX. (d): n = 4 and 3 mice in control and Sdf1^ΔPLT/ΔPLT mice (sham), n = 5 mice in both control and Sdf1^Δ/Δ mice (PNX). (e): n = 5 and 4 mice in control and Sdf1^ΔPLT/ΔPLT mouse groups (sham), n = 4 and 5 mice in pneumonectomized control and Sdf1^Δ/Δ mice groups. Scale bar = 50 μm.

f-i) Proliferation of AEC2s and PCECs in Sdf1^ΔPLT/ΔPLT and control mice after PNX. Expansion of AEC2s and PCECs at day 7 after PNX was determined by immunostaining (f, g) and flow cytometry (h, i), respectively; n = 4 mice per group in (f) and (i); P = 0.001 (f) and 0.0011 (i) between two genotypes. Scale bar = 50 μm.

j) Restoration of AEC1s in Sdf1^ΔPLT/ΔPLT and control mice after PNX. Scale bar= 50 μm.

k) After PNX, activated platelets adhere to PCECs and supply SDF1 to drive regenerative alveolarization. Error bar denotes s.e.m., and line describes mean for panels c, d, e, i. Unpaired two-tail t-test was used to determine the difference between individual groups.

Intravascular infusion of SDF1⁺ platelets rescues impaired alveolar regeneration in Thpo^−/− mice

The importance of platelets in evoking lung regeneration led us to postulate that infusion of platelets into Thpo^−/− mice would rescue the defective regeneration. To this end, we adopted a platelet-infusion model²⁶ to intravenously transfuse platelets into mice (Fig. 4a). After jugular vein infusion, platelets preferentially accumulated in the lungs of Thpo^−/− mice after PNX (Fig. 4b, c). As such, this platelet infusion strategy allows for “gain of function” study to interrogate the influence of platelets on lung alveolar regeneration.

Figure 4. Intravascular transfusion of Sdf1^+/+ but not Sdf1-deficient (Sdf1^−/−) platelets promotes alveolar regeneration in Thpo^−/− mice.

a-c) Platelets accumulate in the lungs of pneumonectomized Thpo^−/− mice after intravascular infusion. Platelets were isolated from β-actin promoter driven-tdTomato (ACTB-tdTomato) mice and infused into Thpo^−/− mice (a). Accumulation of tdTomato⁺CD41⁺ platelets in the lungs of Thpo^−/− mice was determined by flow cytometry and immunostaining; Scale bar = 50 μm.

d-g) Strategy to examine the influence of platelet-derived SDF1 on lung alveolar regeneration. Sdf1^+/+ and Sdf1^−/− platelets were isolated from wild type and Sdf1^Δ/Δ mice and infused into pneumonectomized Thpo^−/− mice, respectively. Recovery of right lung volume (e), mass (f) and respiratory function (g) were determined in recipient mice. In panels (e) and (f), n =5 mice in all groups; P = 0.011 (e) and 0.026 (f) between mice transplanted with Sdf1^+/+ and Sdf1^−/− platelets. In panel (g), n = 6 mice (Sdf1^+/+) and 4 mice (Sdf1^−/−) . P = 0.0013 (left) and 0.0042 (right) between two groups.

h-k) Expansion of AEC2s (h, j) and PCECs (i, k) in Thpo^−/− mice after infusion of Sdf1^+/+ and Sdf1^−/− platelets. Representative immunostaining image of two transplanted groups and flow cytometry graph are shown; n = 5 mixw in all groups; P = 0.00011 (j) and 0.00081 (k) between two platelet types. Scale bar = 50 μm.

l) AEC1s in Thpo^−/− mice following PNX and Sdf1^+/+ and Sdf1^−/− platelet transplantation. Scale bar = 50 μm.

m-o) Alveolar architecture of Thpo^−/− mice receiving platelet infusion after PNX. Alveolar morphology was assessed by H&E staining (m), and alveolar mean linear intercept (n) and number (o) were compared; n = 5 mice in both groups; P = 0.027 (n) and 0.0015 (o) between mice transplanted with Sdf1^+/+ and Sdf1^−/− platelets. Scale bar = 50 um. Error bar defines s.e.m., and line represents mean for panels e, f, g, j, k, n, o. Statistical difference between groups was assessed by unpaired two tail t-test.

We then compared the effects of Sdf1^+/+ and Sdf1-deficient (Sdf1^−/−) platelets on the impaired lung regeneration in Thpo^−/− mice (Fig. 4d). Sdf1^−/− and Sdf1^+/+ platelets were isolated from Sdf1^ΔPLT/ΔPLT and wild type littermates and infused into pneumonectomized Thpo^−/− mice (suppl. Fig. 5). While after PNX similar numbers of infused platelets adhered to PCECs, infusion of Sdf1^+/+ but not Sdf1^−/− platelets into Thpo^−/− mice rescued the restoration of lung mass and volume (Fig. 4e-f), pulmonary function (Fig. 4g), as well as expansion of AECs and PCECs (Fig. 4h-l). Consequently, Sdf1^+/+ platelets infusion to Thpo^−/− mice induced significantly elevated neo-alveologenesis than did Sdf1^−/− platelet transplantation, as evidenced by higher alveolar number and lower mean linear intercept (Fig. 4m-n). Thus, increased bioavailability of SDF1 from the infused platelets rescued the defective neo-alveolarization in pneumonectomized Thpo^−/− mice.

Platelet-derived SDF1 induces membrane MMP14 in PCECs to release EGF-like ligand, stimulating neo-alveologenesis

Induction of membrane MMP14 in PCECs by PNX causes release of EGF-like ligands such as HB-EGF²¹ to stimulate the propagation of adjacent AEC2s. Close association between PCECs and recruited platelets after PNX suggests that platelets stimulate alveolar regeneration by priming the pro-regenerative function of PCEC niche^{1, 14, 15, 24, 54}. Indeed, PCEC expression of MMP14 in Sdf1^ΔPLT/ΔPLT mice was significantly lower than that of control mice after PNX (Fig. 5a). Thus, we analyzed how platelet SDF1 regulates PCEC deployment of MMP14. Supernatant of ADP-stimulated Sdf1^+/+ but not Sdf1^−/− platelets caused membrane MMP14 upregulation in PCECs, which was blunted by PI3K inhibitor LY294002 (Fig. 5b-c). MMP14 was similarly upregulated in both mouse and human PCECs after stimulation of recombinant SDF1, and this upregulation was abrogated by genetic silencing of SDF1 receptors Cxcr4 and Cxcr7 in PCECs (Fig. 5d-f). These data imply that platelets release SDF1 to induce pro-regenerative MMP14 in PCEC niche, igniting regeneration without causing fibrosis (suppl. Fig. 5a).

Figure 5. Platelet-derived SDF1 stimulates Akt pathway to deploy membrane-type MMP14 in PCECs, leading to release of heparin-binding epidermal growth factor (HB-EGF).

a) Expression of MMP14 in Sdf1^ΔPLT/ΔPLT and control mice after PNX. MMP14 was co-stained with PCEC marker VE-cadherin in lung cryosections. Scale bar = 50 μm.

b-c) MMP14 protein level in VE-cadherin⁺ mouse PCECs after treatment with supernatant from indicated platelet types. The effect of PI3K inhibitor LY294002 was also tested. (c): n = 4 cell samples in all groups. P = 0.00028 between PCECs treated with supernatant of Sdf1^+/+ and Sdf1^−/− platelets. P = 0.00011 between PCECs with or without LY294002.

d-f) Protein level of MMP14 was measured in SDF1-stimulated PCECs. Cxcr4 and Cxcr7 were silenced in PCECs by shRNA (shCxcr4 and shCxcr7) and compared with Scrambled (Srb) transduced PCECs (Dashed line). (f): n = 4 cell samples in all groups. P < 0.01 in all shCxcr4 and shCxcr7 groups compared to Srb group (f).

g) Akt activation in PCECs of WT, Thpo^−/−, and Thpo^−/− mice received platelets (+ PLT) after PNX. Level of phosphorylated Akt (pAkt) in isolated PCECs was detected by immunoblot. Protein levels of total Akt and β-actin served to control the amounts of loaded protein.

h-i) PCEC Akt activation in indicated mouse groups after infusion of Sdf1^+/+ and Sdf1^−/− platelets. (i): n = 4 mice in all groups. P = 0.001 between groups infused with Sdf1^+/+ and Sdf1^−/− platelets.

j) Cellular localization of MMP14 in stimulated mouse PCECs was determined by co-staining with junction protein VE-cadherin. Scale bar = 50 μm.

k-l) Membrane level of MMP14 protein in PCECs of Thpo^−/− and WT mice after PNX. Mouse PCEC membrane proteins were biotinylated and isolated (k). MMP14 level was measured in membrane proteins by immunoblot (l). Endothelial-specific surface marker VE-cadherin served as protein loading control. Effect of thrombopoietin (TPO) injection on Thpo^−/− mice was tested.

m-p) Protein levels of PCEC membrane MMP14 (m, o) and HB-EGF in bronchioalveolar lavage fluid (BALF) (n, p) in pneumonectomized mice after platelet infusion. β-actin protein of cells in BALF was utilized for normalization of HB-EGF level. (p), n = 5 mice per group in (o) and (p). P = 0.0012 (o) and 0.0014 (p) between two platelet types. In the presented immunoblot image, each lane indicates individual mouse or cell sample. Error bar depicts s.e.m., and line represents mean for panels c, f, i, o, p. Statistical difference between groups was assessed by unpaired two tail t-test.

Blocking induction of endothelial MMP14 by PI3K inhibitor raised the hypothesis that after PNX platelets deploy SDF1 to stimulate Akt-dependent MMP14 upregulation. To test this notion, we measured the Akt activation/phosphorylation in PCECs of pneumonectomized WT and Thpo^−/− mice (Fig. 5g). PNX caused a time-dependent Akt phosphorylation in the PCECs of WT mice. By contrast, this PCEC Akt activation by PNX was significantly lower in Thpo^−/− mice, which was enhanced by infusion of Sdf1^+/+ rather than Sdf1^−/− platelets (Fig. 5h, i). These findings suggest that deposition of SDF1 by platelets stimulates Akt-dependent upregulation of MMP14 in PCECs after PNX.

Cellular surface localization of MMP14 is critical for the protease activity of membrane-type MMP14^{19, 21, 22}. Treatment of PCECs with supernatant from activated platelets stimulated SDF1-dependent membrane localization of MMP14 protein (Fig. 5j). To determine the distribution of MMP14 in regenerating PCECs, we employed an “in situ” pulmonary vascular perfusion system to selectively biotinylate and fractionate PCEC membrane proteins (Fig. 5k, suppl. Fig. 6a)⁵⁵. At day 7 after PNX, MMP14 level in the isolated PCEC membrane proteins was significantly higher in the pneumonectomized lungs of WT but not Thpo^−/− mice (Fig. 5l). Both TPO injection and transplantation of WT platelets to Thpo^−/− mice augmented membrane enrichment of MMP14 in PCECs (Fig. 5m). Subsequently, release of epithelial-active substrate of MMP14, HB-EGF, in the alveolar space was augmented in Thpo^−/− mice transplanted with Sdf1^+/+ but not Sdf1^−/− platelets, as evidenced by elevated HB-EGF protein level in bronchioalveolar lavage fluid (BALF) (Fig. 5n-p). These findings implicate that SDF1 from activated platelets prime PCEC niche to deploy membrane MMP14, initiating propagation of adjacent AECs.

Inducible endothelial-specific deletion of Mmp14 in mice blocked neo-alveologenesis after PNX

We then employed an EC-specific genetic deletion strategy to assess the contribution of endothelial-derived MMP14 in stimulating neo-alveologenesis. Tamoxifen-inducible EC-specific mouse deleter, VE-Cad-Cre^ERT2/Cdh5(PAC)-Cre^ERT2 mice⁵⁶ were bred with mice harboring LoxP site-flanked Mmp14²⁰. Intraperitoneal injection of tamoxifen to resultant offsprings induced EC-specific Mmp14 ablation (Mmp14^iΔEC/iΔEC) in adult mice (Fig. 6a, suppl. Fig. 6c-d). After PNX, restoration of lung mass and volume and cellular propagation were drastically reduced in Mmp14^iΔEC/iΔEC, compared with control Mmp14^iΔEC/+ mice (Fig. 6b-f). Notably, infusion of platelets failed to rescue the impaired functional alveolar regrowth in Mmp14^iΔEC/iΔEC mice after PNX (Fig. 6g-k). By contrast, injection of MMP14 downstream effector recombinant EGF to Thpo^−/− mice rescued the defective lung regeneration after PNX (Fig. 6l-p, suppl. Fig. 6e-f). These data suggest that recruited platelets after PNX deploy MMP14 in PCEC niche to stimulate EGFR signaling in proximal AEC2s and instigate their expansion, driving neo-alveologenesis and restoration of pulmonary function (Fig. 6q).

Figure 6. Expression of MMP14 in PCEC niche is essential for evoking functional recovery of AECs after PNX.

a) Endothelial cell (EC)-specific inducible deletion of Mmp14 in adult mice. Mice carrying EC-specific promoter VE-cadherin-driven tamoxifen-responsive Cre (Cdh5-PAC-Cre^ERT2) were bred with mice harboring LoxP site-flanked Mmp14 to generate mice with EC-specific deletion of Mmp14 (Mmp14^iΔEC/iΔEC). Mmp14^iΔEC/+ mice harboring EC-specific Mmp14 haplodeficiency were used as control.

b, c) Lung mass (b) and volume (c) in Mmp14^iΔEC/iΔEC and control mice after sham and PNX. n =3 mice in all groups.

d-f) Proliferation of AEC2s (d) and PCECs (f) in Mmp14^iΔEC/iΔEC and control mice. (e): n = 3 animals in all groups; P = 0.0029 (AEC2 proliferation, top panel) and 0.88 (PCEC proliferation, bottom panel) between Mmp14^iΔEC/iΔEC and control group; Scale bar = 50 μm.

g-j) Effect of platelet infusion on the alveolarization in Mmp14^iΔEC/iΔEC mice. Platelets were infused into pneumonectomized Mmp14^iΔEC/iΔEC and compared with control mice (g). Recovery of pulmonary respiratory function (h, i) and AECs (j) was determined. Platelet infusion failed to rescue the defective alveolar regeneration in Mmp14^iΔEC/iΔEC mice. n = 3 mice per group in (h) and (i); P = 0.003 (h), = 0.011 (i) between control and Mmp14^iΔEC/iΔEC mice, Scale bar = 50 μm.

k-o) Influence of recombinant epidermal growth factor (EGF) on Thpo^−/− mice after PNX. EGF was injected pneumonectomized Thpo^−/− mice (k). Proliferation of AEC2s (l) and PCECs (m), recovery of AEC1s (n), and blood oxygenation (o) were assessed. (o) n = 4 mice in both groups; P = 0.006 between two EGF and vehicle injected groups; Scale bar = 50 μm. Error bar defines s.e.m., and line depicts mean for panels b, c, e, h, i, o. Unpaired two tail t-test was used to determine statistical difference between groups.

p) Activated platelets via depositing SDF1 activate MMP14 pathway in PCEC, functionalizing a hemo-vascular niche promoting lung neo-alveologenesis. Upon PNX, activated platelets supply SDF1 to deploy MMP14 in PCEC niche and alveologenic ligand HB-EGF, eliciting propagation of AEC2s and driving alveologenesis.

SDF1 receptors CXCR4 and CXCR7 are required for induction of alveologenic MMP14 in PCECs

To examine the molecular mechanism whereby platelet SDF1 activates PCEC niche, we tested the contribution of SDF1 receptors CXCR4 and CXCR7 on PCECs after PNX. Injection of CXCR4 antagonist AMD3100 blocked cell proliferation and lung function recovery following PNX (suppl. Fig. 7), suggesting the importance of CXCR4 in promoting PNX-induced alveolar regrowth. To circumvent the vascular defect and embryonic lethality caused by Cxcr4 genetic deletion⁴⁸, we inducibly deleted Cxcr4 and Cxcr7 in the ECs of adult mice (Cxcr4^iΔEC/iΔEC and Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC) (Fig. 7a, suppl. Fig. 8a-b). Alveolar regeneration in Cxcr4^iΔEC/iΔEC mice was significantly impaired compared to control Cxcr4^iΔEC/+ mice, including expansion of AEC and PCEC (Fig. 7b-e), recovery of lung function (Fig. 7f-g), MMP14 induction in PCECs (Fig. 7h), and restoration of mass and volume (Fig. 7i-j). Of note, this defect in functional alveolar regrowth was further exacerbated in Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice. Impaired lung regeneration in Cxcr4^iΔEC/iΔEC and Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice was associated with attenuation of Akt activation in PCECs (Fig. 7k, suppl. Fig. 8c). To illustrate the influence of platelets on SDF1-mediated signaling in PCEC niche, we infused platelets to Cxcr4^iΔEC/iΔEC and Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice and tested the regenerative response (Fig. 7l). Infusion of SDF1⁺ platelets into mice did not restore the alveolar release of HB-EGF in either Cxcr4^iΔEC/iΔEC or Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice (Fig. 7m-n). Taken together, these findings suggest that platelets deploy SDF1 to trigger CXCR4 and CXCR7 signaling in PCECs, enabling a pro-regenerative niche that drives regenerative alveolarization after PNX.

Figure 7. Platelets activate CXCR4 and CXCR7 in PCEC niche to drive alveolar regeneration.

a) Endothelial cell (EC)-specific inducible deletion of Cxcr4 and Cxcr7 in adult mice. VE-cadherin-Cre^ERT2/Cdh5-PAC-Cre^ERT2 mice were crossed with floxed Cxcr4 and Cxcr7 mice to induce EC-specific deletion of Cxcr4 (Cxcr4^iΔEC/iΔEC) and both Cxcr4 and Cxcr7 (Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC) in adult mice. Cxcr4^iΔEC/+ mice was used as control.

b-e) Proliferation rate of AEC2s and PCECs in control, Cxcr4^iΔEC/iΔEC, and Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice after PNX. n = 4 mice in both control and Cxcr4^iΔEC/iΔEC group, and n = 3 mice in Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC group; Scale bar = 50 μm. In panel (c), P = 0.0066 (control versus Cxcr4^iΔEC/iΔEC) and 0.0015 (control versus Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC). In panel (d), P = 0.0013 (control versus Cxcr4^iΔEC/iΔEC) and 0.00055 (control versus Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC).

f-g) Functional alveolar regrowth in indicated mouse groups following PNX. In panels (f) (g), n = 3 mice in both control and Cxcr4^iΔEC/iΔEC group; n = 4 mice in Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC group. P = 0.00069 (f) and 0.014 (g) between control and Cxcr4^iΔEC/iΔEC mice. P = 0.00026 (f) and 0.0017 (g) between control and Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC group.

h) PCEC MMP14 protein level in control, Cxcr4^iΔEC/iΔEC, and Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice_was tested by flow cytometry.

i-k) Weight (i), volume (j) and PCEC Akt activation (k) in right lungs after PNX. Western blot was used to test p-Akt level in PCECs. n = 5, 4, 4, 5, 4, 4 mice (i) and = 5, 4, 4, 3, 5, 4 animals (j) in control Cxcr4^iΔEC/+, Cxcr4^iΔEC/iΔEC, Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC groups that underwent sham and PNX, respectively.

l-n) BALF level of HB-EGF in pneumonectomized Cxcr4^iΔEC/iΔEC and Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice after Infusion of Sdf1^+/+ platelet. Influence of platelets on alveolar regeneration of indicated mouse groups was performed as described in (l), HB-EGF in BALF was tested by Western blot (m) and quantified (n). In (n), n = 5 mice in control Cxcr4^iΔEC/+ group, n= 4 mice in other shown groups. In the shown immunoblot image, each lane indicates sample from individual mouse. Error bar denotes s.e.m., and line defines mean for panels c, d, f, g, i, j, n. Statistical difference between groups was analyzed by unpaired two tail t-test.

Modulation of SDF1 signaling in the pulmonary vascular niche by VEGFR2/FGFR1 pathway

Activation of PCECs during alveolarization is a coordinated process that requires synergistic effects of various signaling molecules, including angiogenic factors VEGF-A and FGFs¹. Indeed, activation of both VEGFR2 and FGFR1 pathways was attenuated in PCECs of Thpo^−/− mice after PNX (Fig. 8a-c). This finding implicates that activation of VEGFR2 and FGFR1 in PCECs after PNX is at least partially dependent on platelets. To test the correlation of SDF1 signaling with VEGFR2/FGFR1 pathway, we tested the expression of CXCR4 in mice with EC-specific deletion of VEGFR2 and FGFR1¹ (Vegfr2^iΔEC/iΔECFgfr1^iΔEC/+) after PNX (Fig. 8d-e). Notably, protein level of CXCR4 in PCECs was significantly upregulated by PNX in control but not Vegfr2^iΔEC/iΔECFgfr1^iΔEC/+ mice. These data suggest that SDF1 signaling (receptor CXCR4) in PCECs is regulated by previously shown pro-regenerative VEGFR2 and FGFR1 pathway after PNX.

Figure 8. Regulation of SDF1 signaling in PCEC niche by VEGFR2/FGFR1 pathway.

a) Strategy to test the activation of VEGFR2, FGFR1 and expression of SDF1 receptor CXCR4 in PCECs of indicated mouse groups.

b-c) Activation of VEGFR2 and FGFR1 in PCECs of Thpo^−/− mice after PNX. Phosphorylation of VEGFR2 (p-VEGFR2) was tested by Western blot. To test FGFR1 activation, phosphorylation of downstream effector FRS2 (p-FRS2) was similarly assessed. Protein levels of VEGFR2 and FRS2 and β-actin were also measured as control. n = 4 mice in all tested groups; P = 0.0172 (VEGFR2 activation) and 0.0153 (FGFR1 activation) between WT and Thpo^−/− mice.

d-e) Expression of CXCR4 protein in mice that are deficient of Vegfr2 and Fgfr1 in ECs (Vegfr2^iΔEC/iΔECFgfr1^iΔEC/+). Vegfr2^iΔEC/iΔECFgfr1^iΔEC/+ mice was generated as previously described¹. n = 4 animals in both groups, and P = 0.0005 between two genotypes.

f-g) Rescue effect of VEGF₁₆₄ and SDF1 in pneumonectomized Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC and Vegfr2^iΔEC/iΔECFgfr1^iΔEC/+ mice, respectively. Restoration of gas exchange function (f) and PCEC Akt activation (g) were tested. P = 0.0319 between Vegfr2^iΔEC/iΔECFgfr1^iΔEC/+ mice injected with SDF1 and vehicle, and P = 0.28 between Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice injected with VEGF₁₆₄ and vehicle; n = 4 mice per group..

h-i) PCEC Akt activation in indicated mice was determined. n = 6 mice in all tested groups. In the presented immunoblot image, each lane indicates individual mouse sample. Error bar stands for s.e.m., and line represents mean for panels c, d, g, i. Statistical difference between groups was determined by unpaired two tail t-test.

j) Regulation of SDF1 signaling in PCEC niche by VEGFR2/FGFR1 pathway after PNX. Activation of VEGFR2 and FGFR1 is mediated by both platelet-dependent and independent mechanisms. Activation of VEGFR2/FGFR1 synergistically acts with SDF1 receptors (CXCR4/CXCR7) on PCECs to trigger vascular niche-mediated neo-alveologenesis.

k) Recruitment of activated platelets via mobilizing SDF1 primes PCEC niche and drives lung alveolar regeneration/regrowth. Upon PNX, activated platelets supply SDF1 to activate CXCR4 and CXCR7 on PCECs. Platelet-mediated CXCR4/7-Akt activation deploys MMP14 in PCEC niche and causes release of alveologenic ligand HB-EGF, eliciting propagation of AEC2s and driving neo-alveologenesis.

We then used Vegfr2^iΔEC/iΔECFgfr1^iΔEC/+ mice and Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice to reveal the functional regulation of SDF1 signaling by VEGFR2/FGFR1 in PCEC niche after PNX (Fig. 8f). First, restoration of lung function (blood oxygenation) was significantly improved in Vegfr2^iΔEC/iΔECFgfr1^iΔEC/+ mice by SDF1 injection after PNX. By contrast, VEGF injection into Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice failed to enhance the recovery of lung function in mice (Fig. 8g). The different effects caused by VEGF-A and SDF1 injection implicate that CXCR4/7-mediated SDF1 signaling in PCECs is downstream of VEGFR2 pathway.

This potentiation of SDF1 signaling by VEGFR2/FGFR1 pathway was further evidenced by the analysis of Akt activation in PCECs (Fig. 8h-i). Compared to WT mice, Akt activation in PCECs was blocked in both Vegfr2^iΔEC/iΔECFgfr1^iΔEC/+ and Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice. Of note, while SDF1 injection to Vegfr2^iΔEC/iΔECFgfr1^iΔEC/+ mice restored PCEC Akt activation, Akt activation in PCECs of Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC mice was not enhanced by VEGF-A injection. Thus, activation of VEGFR2 and FGFR1 promotes SDF1-CXCR4/7 signaling to stimulate activation of PCEC niche, driving regenerative alveolarization process (Fig. 8j), and it's plausible that after PNX, platelet-derived SDF1 co-operates with other pathways to synergistically prime pro-regenerative PCECs, initiating alveolar regeneration (Fig. 8k)^{29, 33, 34.}

Discussion

Here, the centrality of platelet-derived SDF1 in inducing alveolar regeneration was revealed by “platelet-specific” gain and loss of function studies: 1) regenerative alveolarization was impaired in thrombocytopenic mice caused by thrombopoietin knockout (Thpo^−/−) and pharmacological platelet depletion (Fig. 1); 2) Platelet-specific and inducible genetic ablation of the Sdf1 gene abrogated lung alveolar regeneration (Fig. 2-3); 3) intravascular transfusion of Sdf1^+/+ platelets, but not Sdf1^−/− platelets, rescued the defective alveolarization of Thpo^−/− mice (Fig. 4, 5). 4) Using EC-specific gene deletion strategy (Mmp14^iΔEC/iΔEC and Cxcr4^iΔEC/iΔECCxcr7^iΔEC/iΔEC), we demonstrate that this rescue effect of SDF1 platelets is mediated via CXCR4/7-dependent deployment of pro-regenerative MMP14 pathway in PCEC membrane (Fig. 6-8). Hence, we propose that PNX recruits SDF1⁺ platelets to prime PCECs in the remaining lungs, functionalizing a hemo-vascular niche to orchestrate functional restoration of pulmonary tissues.

How does the loss of left lung recruit and activate platelets in the right lungs remains to be studied. It is plausible that after PNX, increased blood flow and circumferential shear force in the right lungs activate platelets to adhere to pulmonary capillaries^{28, 31, 32, 36}. Alternatively, perturbation of biomechanical forces within the remaining lungs activates pulmonary capillaries, thereby recruiting and activating circulating platelets⁴². In addition, how activated platelets influence other types of lung progenitor cells needs to be investigated in the future^{10, 11, 13, 57}.

Of note, the morphology of AEC1s is compromised when regeneration fails. After PNX, the loss of left lung lobe caused significantly increased mechanical stretch in the remaining lungs, possibly stimulating the matrix remodeling process⁶. Conceivably, in WT mice with normal regenerative capacity, this remodeling process coordinately accompanies the expansion of AEC1s. By contrast, when regeneration/cellular propagation fails in platelet-depleted mice or mice with defective vascular niche, the alveolus undergoes remodeling in the absence of concomitant expansion of AECs. As such, in these mutant or antibody-treated mice, discordance between alveolar remodeling and lack of concurrent AEC propagation not only prevented the growth of new alveoli but also disrupted the morphology of existent AECs, compromising alveolar architecture.

Here we have utilized cell-type specific “loss and gain of function” studies to demonstrate that recruited platelets release SDF1 to activate CXCR4/CXCR7 on PCECs at early phase after PNX (day 1-5), eliciting cellular propagation and alveolarization that peaks at day 7. It is likely that platelets represent a subpopulation of bone marrow-derived cells recruited into the lungs of pneumonectomized mice⁴⁹. Whether bone marrow-derived cells facilitate the pulmonary recruitment of activated platelets after PNX remains to be investigated. Furthermore, SDF1 from other niche cells^50-53 could play a more prominent role in modulating tissue repair in other injury models.

We have also reconciled the mechanistic link between previously demonstrated VEGFR2-FGFR1 pathway^{1, 58} and the presented endothelial SDF1-CXCR4/7 interaction²³ in the study. SDF1-CXCR4 signaling is regulated by VEGFR2/FGFR1 pathway in PCECs after PNX, and Thpo^−/− mice also exhibited blocked VEGFR2/FGFR1 signaling in PCECs. These findings implicate that platelets also contribute to activation of VEGFR2/FGFR1 pathway that further potentiates SDF1 signaling in PCECs. It's likely that activation of both pathways in PCECs leads to MMP14-dependent lung alveolarization, which is initially stimulated by platelets after PNX.

Lung disease remains worldwide leading cause of death for adults^{7, 14, 59-62}. Damage to the lungs after trauma or chemical injury results in initiation of reparative pathways. However, in most instances lung repair is insufficient to overcome the loss of functional parenchymal tissues^{12, 63-65}. Our finding implicates that restoration of functional lung mass could be elicited in adult mammals by a coordinated interaction between alveolar progenitor cells and pro-regenerative cues from vascular niche. As such, properly stimulating the cellular crosstalk between alveolar progenitor cells with surrounding niche^{5, 8, 9, 13, 66, 67} could instigate regeneration in adult human lungs with limited regenerative capacity, implicating the translational value of our study in combating chronic destructive lung diseases.

Uncovering the effects of platelet-derived SDF1 on inducing lung regeneration holds additional clinical implication. First, it's conceivable that thrombocytopenic patients will exhibit impaired regenerative ability after lung injury and show poor prognosis. To this end, periodic administration of SDF1 or EGF into thrombocytopenic patients (e.g. at intensive care unit) could stimulate functional lung repair. Furthermore, the finding that infusion of SDF1⁺ platelets initiates and sustains functional alveolarization suggests that transplantation of properly primed platelets represents effective cell therapy approaches to enable lung regeneration and repair.

Taken together, we have revealed a paradigm that loss of left lung lobe recruits platelets to deploy SDF1, preconditioning pro-regenerative PCEC niche and eliciting lung alveolar regeneration. Infusion of functional platelets rescued the impaired lung regeneration by re-establishing inductive endothelial niche. Unraveling the cellular and molecular basis whereby platelets orchestrate lung regeneration permits a clinical strategy to initiate regenerative approach for lung diseases.

Methods

Animals

Cdh5(PAC)-Cre^ERT2 (VE-cadherin-Cre^ERT2) mouse line was provided by Dr. Ralf Adams⁵⁴, Rosa-Cre^ERT2 animals expressing tamoxifen-responsive inducible Cre^ERT2, loxP sites flanked Cxcr4 and Cxcr7 mice, and Thpo^−/− mice were described previously^{1, 27, 43}. Floxed Mmp14 mice were kindly offered by Dr. Steve Weiss at University of Michigan¹⁷. C57/B6, Pf4-Cre, floxed Sdf1⁴⁸, and actin-tdTomato mice ubiquitously expressing tdTomato fluorescent protein (β-actin promoter-driven tdTomato) were obtained from Jackson laboratory (Bar Harbor, Maine). To active Cdh5(PAC)-Cre^ERT2 and Rosa-Cre ^ERT2, mice were treated with tamoxifen for six times at dose of 150 mg/kg. After daily injection of tamoxifen for three consecutive days, mice were rested for three days and then treated with tamoxifen for another three days. Mice were utilized for surgery or analysis at least 30 days after final tamoxifen treatment. To compare phenotypes between different genotypes, age/sex/weight matched littermates were utilized in corresponding experimental groups. Mice at 20-28 grams of bodyweight were used. Investigators that performed mouse lung regeneration model and analyzed the pattern and extent of cell activation/proliferation were randomly assigned with animals or samples from different experimental groups and were blind to genotype of individual groups. All animal procedures were under the guidance of NIH and approved by IACUC at Weill Cornell. Chemicals were from Sigma (St Louis, MO) unless specified otherwise.

Left lung unilateral pneumonectomy (PNX) model and measurement of alveolar regeneration

PNX procedure was adapted as described¹. Mice were anaesthetized by 100 mg/kg intraperitoneal ketamine and 10 mg/kg xylazine. Orotracheal intubation was performed in anesthetized and mechanically ventilated mice. Left lung lobe was resected with a suture tied around the hilum. Sham-operated mice were subjected to thoracotomy without lobe resection. To determine the role of recombinant EGF in alveolar regeneration, Mice were injected with 500 μg/kg recombinant mouse EGF (Abcam) on daily basis after PNX.

Immunostaining and morphometric analysis

Each parameter from individual animal was measured at least twice and averaged. Before sacrificing, mice were anaesthetized, blood was obtained via the inferior vena cava, and the remaining right lung lobes received intratracheal instillation of reconstituted OCT (Tissue-Tek^®) to a pressure of 25 cm H₂O. The trachea was then tied under pressure, and the lungs were cryoprotected and snap frozen in OCT. For immunofluorescence microscopy, the tissue sections (10 μm) were blocked with 5% normal donkey serum/1%bovine serum albumin/0.1% Triton X-100 and incubated in primary antibodies. Pulmonary capillary endothelial cells (PCECs) were identified by staining of anti-VE-cadherin polyclonal Ab (pAb, R&D Systems AF1002, 2 μg/ml), type I alveolar epithelial cells (AEC1s) and type II alveolar epithelial cells (AEC2s) were determined by staining of podoplanin (R&D Systems, AF3244, 2 μg/ml) and aquaporin-5 (Abcam, ab78486, 2 μg/ml) for AEC1s and pro-surfactant protein c (SFTPC) (Millipore, Ab3786, 2 μg/ml) for AEC2s, respectively. Platelet deposition was detected using anti-CD41 monoclonal antibody (MWReg30, BD Biosciences, 1 μg/ml). After incubation in fluorophore-conjugated secondary antibodies (2.5 μg/ml, Jackson ImmunoResearch), nuclear counterstaining was performed with DAPI (Invitrogen). No appreciable staining was observed in isotype IgG controls. AEC2s and PCECs were quantified by staining with antibodies recognizing SFTPC and VE-cadherin, respectively. Fluorescent images were captured on AxioVert LSM710 microscope (Zeiss). For image analysis, staining signal in each slide (such as SFTPC, VE-cadherin, podoplanin, aquaporin-5, and CD41) were independently evaluated and quantified by two investigators from five randomly selected fields of view. Analysis of the image was performed blindedly. Investigators were not aware of the genotype of animals or identity of samples during scoring.

Lung cell proliferation in vivo was measured by BrdU uptake. Mice received a single intraperitoneal injection of BrdU (Sigma) (at a dose of 50 mg/kg animal weight) 60 min before sacrificing and the incorporation of BrdU was measured by immunostaining on cryosections and flow cytometry as previously descrived^{1, 51}. Cryosections were stained using the BrdU Detection System (BD Biosciences) and fluorophore-conjugated secondary antibodies (2.5 μg/ml, Jackson ImmunoResearch)^{1, 51}. Extent of BrdU incorporation was first determined in sham operated mice of all used genotypes. To assess the difference in cell proliferation after PNX, the percentage of BrdU⁺VE-cadherin⁺ proliferating PCECs and BrdU⁺SFTPC⁺ AEC2s in both control and mutant groups were compared at day 7.

Measurement of functional alveologenesis after PNX

Lung respiratory function parameters, including inspiratory capacity and lung parenchymal tissue compliance, were measured using the forced oscillation technique operated by flexiWare 7 software in a computer-controlled piston ventilator (SCIREQ Inc). Inspiratory capacity was determined between the plateau pressure measurements of the lung capacity and functional residual volume. Oxygen tension in the arterial blood was measured as previously described using I-Stat⁶⁸ (Abbott Laboratories, Abbott Park, IL).

Hematoxylin and eosin (H&E) staining was performed on retrieved lung tissues to evaluate alveologenesis after PNX. Alveolar structure in each H&E slide was independently quantified by two investigators from five random fields. Mean linear interception (Lm) was measured as previously described⁶⁹. Briefly, 10 equally distributed horizontal lines and 11 evenly distributed vertical lines were drawn over an H&E-stained section. For each line, the intercepts with the alveolar tissue are counted. Lm is calculated as the averaged ratio between line length and the number of intercepts placed on the lung section.

Alveolar number was determined as previously described⁷⁰. Briefly, the lungs were cut into 3-mm thick lung slices and subsequent cutting of 3-mm wide bars with randomized tissue orientation. Embedded bars were cut into 20-μm thick serial sections. Sections were collected on slides and subjected to H&E staining for determination of a mean bar thickness. The block was then cut to a series of 5-μm thick serial sections and collected on slides. The ratio of serial section thickness/mean bar thickness was then determined. To obtain slide for alveolar number determination, a series of sections per tissue block were systematically scanned along the x- and y-axis to yield a uniform, random sample with disector pairs of 600 μm × 600 μm sections. Then the number of alveoli was measured by assessing the Euler number (x) of the network of alveolar openings within a counting frame. For measurement of Euler number, all alveolar openings that in the examined section were counted in each frame. To increase the efficiency of alveolar number measurement, both directions in the frame were measured in individual field of view. Then the total number of alveoli per lung was calculated based on alveolar number within the area of counting frame, disector pair size on slide, as well as the ratio between serial section thickness and mean bar thickness.

Isolation and analysis of mouse PCECs

Isolation of PCECs and examination of phosphorylation and total protein level of Akt, VEGFR2, FRS2, CXCR4, and MMP14 were carried out as described^{1, 51}. Briefly, the lungs was perfused at 5 ml/min through the pulmonary artery with Liver Perfusion Medium (Invitrogen) at 37 °C for 10 min, followed by perfusion with Liver Digest Medium (Invitrogen) for another 10 min. 1 ml digestion medium was also injected into the alveolar space via exposed trachea. The lung tissue was then dissociated in Hepatocyte Wash medium (Invitrogen), passed through dacron fabric with 70-μm pores and the flow through containing PCECs was collected, washed twice, and pelleted at 350g for 7 min. Fractions containing PCECs were enriched, washed with an equal volume of PBS and centrifuged at 900g for 7 min. The cell pellet was then washed with PBS at 350 g for 7 min and incubated with Dynabeads® Magnetic beads (Invitrogen) coated with rat anti-mouse CD31 and ICAM-2 antibodies (MEC13.3, BD Biosciences). The purification of CD31⁺ICAM2⁺ PCECs was carried out following the manufacturer's protocol. To maintain protein phosphorylation in the isolated PCECs, mouse lungs were perfused via pulmonary artery with Phosphatase Inhibitor Cocktails (Sigma) after mice were sacrificed, and whole processing was carried out in the presence of phosphatase inhibitor cocktails.

Isolated platelets or cultured PCECs were lysed with RIPA (radio-immunoprecipitation assay) buffer: 1X TBS (tris-buffered saline), 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (v/v) SDS (sodium dodecyl sulfate), 1mM sodium orthovanadate, 10mM NaF, and 1X protease inhibitor cocktail (Roche). Phosphorylation and protein level of VEGFR2 and FGFR1 downstream effector FRS2 in PCECs was carried out as described⁵¹.

Equal amounts of protein were subjected to SDS-PAGE (SDS-polyacrylamide gel electrophoresis) in 4-12% Bis-Tris gels (Invitrogen). Proteins were transferred to nitrocellulose membranes, and underwent immunoblotting with antibodies against VE-Cadherin (R&D, AF1002, 1 μg/ml), VEGFR2 (Cell Signaling, 2472, 1 μg/ml), phosphorylated VEGFR2 (Cell Signaling, 3817, 1 μg/ml), total AKT (Cell Signaling, 9272, 1 μg/ml), phosphorylated Akt (Cell Signaling, 2965, 1 μg/ml), total FRS-2 (Abcam, ab137458, 1 μg/ml), total CXCR4 (Abcam, ab2074, 2 μg/ml), total MMP14 (Abcam, ab51074 and ab53712, 1 μg/ml). SDF1 (Cell signaling, 3740, 2 μg/ml). β-actin (Santa Cruz, sc-1616, 2 μg/ml) in the loaded sample was also tested to control for the loaded protein amount.

To test the protein components in the alveolar space, bronchioalveolar lavage fluid (BALF) was obtained from sham or pneumonectomized mice by lavaging 1 ml of PBS through the trachea, and retrieved BALF was concentrated to 100 μl with protein concentration column (Millipore). Heparin binding epidermal growth factor (HB-EGF) protein level in BALF was assessed as previously described¹, using rabbit polyclonal antibody from Santa Cruz.

Depletion of circulating platelets, stimulation of thrombopoiesis

To stimulate thromobopoiesis, recombinant thrombopoietin (TPO) was intraperitoneally injected into Thpo^−/− or WT mice at dose of 25 μg/kg on daily basis ten days before PNX and afterwards. To deplete circulating platelets, rat anti-mouse CD41 monoclonal antibody (BD Biosciences, clone MWReg30) was intraperitoneally injected into mice at dose of 10 mg/kg every three days. Recombinant TPO, EGF, and SDF1 were obtained from Peprotech. Vehicle for individual cytokine was also injected as control group.

The effects of injected cytokine on alveolar regeneration were evaluated with control groups treated with vehicle, including alteration in circulating platelets and parameters of alveologenesis (as described above). In particular, mouse platelets were collected at indicated time points from the retrobulbar venous plexus of anesthetized mice, using heparinized hematocrit tubing. Platelet count in peripheral blood was then determined with automated Advia-120 Hematology System.

Isolation and stimulation of mouse platelets and intravascular infusion of platelets into mice

In order to maximize the yield of platelets, the blood remaining from this first centrifugation was diluted with PBS to the original volume and re-centrifuged. Isolated mouse platelets by centrifugation were treated with agonist ADP (5 or 10 μM) or 0.1 U/mL thrombin. After agonist addition, reaction was terminated and incubation microfuge tubes were centrifuged (1 minute, 14,000 g). Supernatant of stimulated platelets was then aliquoted for activation of primary PCECs.

Strategy to infuse platelets via jugular vein was adopted from previously described⁴⁹. Platelets were isolated and concentrated from mice with inducible deletion of Sdf1 (Sdf1^Δ/Δ mice) to obtain Sdf1^−/− platelets. Platelets from their wild type littermate, or β-actin promoter-driven tdTomato reporter mice were utilized as Sdf1^+/+ platelets. After isolation, 2×10⁹ Sdf1^+/+ or Sdf1^−/− platelets were infused through exposed jugular vein of mice over a period of 15 minutes. This injection route yielded the highest accumulation of platelets in the pulmonary vasculature of recipient mice. To track the localization of infused CD41⁺tdTomato⁺ platelets in the recipient mouse lungs, total cells were isolated from recipient and analyzed for surface expression of tdTomato fluorescent signal and CD41. Immunofluorescent staining of CD41 and VE-cadherin was also performed to examine the association of PCECs by transplanted platelets, as described in “Immunostaining and morphometric analysis”.

Cultivation of mouse and human PCECs and gene knockdown

Primary mouse PCECs were obtained from Angiocrine Bioscience (New York, NY) and cultured as previously described¹. Human PCECs were from ScienCell Research Laboratories and cultivated following vendor's instruction. To knockdown Cxcr4 and Cxcr7 selectively in LSECs, Cxcr4 and Cxcr7/Scrambled short hairpin RNA (shRNA) lentiviruses were generated by co-transfecting 15 μg of shuttle lentiviral vector containing Id1/Scrambled shRNA, 3 μg of pENV/VSV-G, 5 μg of pRRE and 2.5 μg of pRSV-REV in 293T cells by Fugene 6 (Roche Applied Science). Viral supernatants were concentrated by ultracentrifugation. These concentrated viral preparations were used to transduce PCECs. Human Cxcr4 was silenced by treatment with combined shRNA clones: TRCN0000004052, TRCN0000004053, TRCN0000004054, and TRCN0000004055 (Open Biosystems), and Cxcr7 was knocked down with clone TL305345 (Origene). Cxcr4 was knocked down in mouse PCECs by transduction with combination of clones TRCN0000028678, TRCN0000028704, TRCN0000028724. Cxcr7 was silenced in mouse PCECs with shRNA clone TL515747 from Origene.

Supernatant from platelets was used to treat PCECs after starvation in minimal media that consists of Media 199 (HyClone) and 2% BSA. Six hours after treatment with platelet supernatant or 10 ng/ml SDF1, PCECs were retrieved for analysis of MMP14 protein level by immunoblot after cell lysis, immunostaining, and flow cytometry. MMP14 was detected by rabbit polyclonal antibody from Abcam (ab51074 and ab53712, 1 μg/ml).

Isolation of membrane protein of PCECs after pulmonary perfusion and in situ biotinylation

Biotinylation of PCEC membrane protein in mice was achieved by pulmonary erfusion via pulmonary artery⁵³. After mouse pulmonary artery was exposed, a 25-gauge catheter was inserted and fastened. PBS was used to perfuse mouse pulmonary vasculature. Then pulmonary vasculature was perfused with PBS containing 10 mg/ml membrane-impermeable sulfo-NHS-LC-Biotin that recognizes primary amine in protein (Pierce) at 1 ml/min for 10 min. PCEC membrane proteins that are accessible to circulation were biotinylated. After unconjugated biotin was quenched by 20 mg glycine, lung tissues were harvested and homogenized, and biotinylated proteins were isolated by 500 μl/g tissue streptavidin beads (Pierce) and subjected to immunoblot against MMP14 and VE-cadherin after sonication and boiling. This in situ biotinylation strategy provides an approach to isolate and assay membrane fractions of lung ECs. Protein level of MMP14 in isolated membrane proteins after PNX was determined by immunoblot (Abcam), and EC-specific marker VE-cadherin was also blotted (R&D systems) as control for loading of endothelial membrane proteins. To control for the purity of isolated EC memebrane proteins, endothelial membrane proteins such as platelet-endothelial cell adhesion molecule (PECAM)-1 (Santa Cruz Biotechnology, sc-1506, 10 μg/ml), intercellular cell adhesion molecule (ICAM)-1 (Santa Cruz Biotechnology, sc-1511, 10 μg/ml), VEGF receptor 2 (VEGFR2), angiotensin converting enzyme (sc-20998, 2 μg/ml), integrin αv (sc-6618, 5 μg/ml), α5 (sc-10729, 5 μg/ml), E-cadherin (sc-7870, 10 μg/ml), and cytoplasmic proteins α-tubulin (sc-53646, 10 μg/ml), and β-actin were also examined in the isolated membrane fractions by immunoblot as described above.

Mouse model of unilateral in situ lung ischemia-reperfusion

Acute lung injury was also introduced by a mouse model of lung ischemia-reperfusion (I/R). Unilateral left lung I/R was created using a protocol previously described in detail⁶⁸. Briefly, after performing a thoracotomy in ventilated anesthetized mice, the hilum of the left lung was cross-clamped for 30 min followed by 120 min of reperfusion. Five minutes prior to sacrifice, 400 U of heparin was injected intravenously to prevent postmortem clotting and the left lobe of the lung was excised. Sham-operated mice underwent the same procedure except that clamping of the hilum was omitted. Lung specimens were weighed and homogenized in PBS (pH 7.2) containing protease inhibitor cocktail (Sigma), aprotinin (30 KIU/ml), heparin 15 U/ml and 100 mM 6-aminohexanoic acid (EACA). Aliquots of tissue homogenates were prepared for analysis. Lung fibrin was extracted from one aliquot, detected by Western blot using fibrin β-chain specific antibody 350 (American Diagnostica, Inc., 5 μg/ml), as described previously ⁶⁸. Total lung proteins were extracted with 1% SDS and 1% Triton X-100 and subjected to Western blot using anti-β-actin. Lung fibrin was quantified by densitometric analysis and normalized to β–actin. Lung fibrin deposition in lungs was compared between different groups.

Flow cytometric analysis

To reveal platelet and PCEC activation, deposition, and proliferation, total lung cells were isolated from perfused mouse lung tissues after dissociation with medium containing collagenase/dispase/DNase¹. Retrieved lung cells were incubated with conjugated antibodies and analyzed by flow cytometry on LSRII-SORP (BD)⁵¹. Data were processed with FACSDiva 6.1 software (BD). All doublets were ruled out by FSC-W × FSC-H and SSC-W × SSC-H analysis. Monoclonal antibodies were conjugated to Alexa Fluor dyes or Qdots per manufacturer's protocols (Molecular Probes/Invitrogen), including VE-cadherin (clone BV13, ImClone, 5 μg/ml); CD41 (clone MWReg30, BD Bioscience, 5 μg/ml), and P-selectin (clone RB40.34, BD Bioscience, 5 μg/ml). Single-stained channels were used for compensation. Flow cytometry analysis was performed using various controls, including unstained cells, isotype antibodies, and fluorophore minus one controls for determining gates and compensations in flow cytometry.

Statistical analysis

Experiments were repeated for at least three times. We included all tested animals for quantification to analyze statistical difference. Representative image from each animal group is presented in the figure. If higher variability was found, we increased the sample size to fully confirm statistical significance. No statistical method was used to predetermine sample size. Statistical analysis between individual experimental groups was determined by unpaired two tail t-test. All data points from individual animal or cell are presented in the format of dot plot.

Reproducibility of Experiments

All presented representative images were obtained from independently repeated experiments. Immunostaining images in Fig. 1f, j, o; 2a, e, j; 3g, j; 4h, l; 5a, i; 6d, k, m, o; 7b; supplementary Fig. 1a-d, supplementary Fig. 2a, c, e; supplementary Fig. 3g, h; supplementary Fig. 5a; supplementary Fig. 7a were independently repeated three times. Immunoblot images in Fig. 2i; 3b; 5b, d, e, g, h, l, m, n; 7k, m; 8b, e, h; supplementary Fig. 6a, c; supplementary Fig. 8a, b were from four repeated experiments, and flow cytometry micrograph in Fig. 1b, h, n; Fig. 2b; 3h; 4i; 6f, n; 7e, h; supplementary Fig. 7c were repeated for three times. Utilized animal numbers per groups are described in corresponding figure legend.