Balanced Wnt/Dickkopf-1 signaling by mesenchymal vascular progenitor cells in the microvascular niche maintains distal lung structure and function

Abstract

The well-described Wnt inhibitor Dickkopf-1 (DKK1) plays a role in angiogenesis as well as in regulation of growth factor signaling cascades in pulmonary remodeling associated with chronic lung diseases (CLDs) including emphysema and fibrosis. However, the specific mechanisms by which DKK1 influences mesenchymal vascular progenitor cells (MVPCs), microvascular endothelial cells (MVECs), and smooth muscle cells (SMCs) within the microvascular niche have not been elucidated. In this study, we show that knockdown of DKK1 in Abcg2^pos lung mouse adult tissue resident MVPCs alters lung stiffness, parenchymal collagen deposition, microvessel muscularization and density as well as loss of tissue structure in response to hypoxia exposure. To complement the in vivo mouse modeling, we also identified cell- or disease-specific responses to DKK1, in primary lung chronic obstructive pulmonary disease (COPD) MVPCs, COPD MVECs, and SMCs, supporting a paradoxical disease-specific response of cells to well-characterized factors. Cell responses to DKK1 were dose dependent and correlated with varying expressions of the DKK1 receptor, CKAP4. These data demonstrate that DKK1 expression is necessary to maintain the microvascular niche whereas its effects are context specific. They also highlight DKK1 as a regulatory candidate to understand the role of Wnt and DKK1 signaling between cells of the microvascular niche during tissue homeostasis and during the development of chronic lung diseases.

INTRODUCTION

The tightly controlled formation of the alveolar-capillary network, and the complex signaling between vasculature, mesenchyme, and epithelium that occurs during lung development provides some insight into how vasculopathy may influence loss of lung structure and function during the development of chronic lung diseases (CLDs) (2, 13, 14, 24, 41, 59, 63, 70). The etiology of microvascular remodeling and mechanisms through which it contributes to the development and severity of various CLDs remain unknown, due in part to a lack of adult rodent models evaluating early-stage vasculopathy. The relevance of vasculopathy to the pathophysiology of CLDs has not been resolved as conflicting evidence depicts angiogenesis as both, reparative or pathologic (10, 12, 25, 33, 35, 36, 46, 51, 57, 61, 65, 68), highlighting the need to identify the early mechanisms underlying the initiation of the disease.

Our previous work has demonstrated that ABCG2-expressing adult mesenchymal vascular progenitor cells (MVPCs) are a key component of lung microvascular homeostasis, angiogenesis in response to injury, and are necessary to maintain distal lung tissue structure (30, 62). MVPC function is dependent on canonical Wnt signaling, with β-catenin as a central mediator (30, 62). The Wnt signaling pathway controls a variety of developmental and biological processes including cell fate, proliferation, and migration, and recent studies have also linked the Wnt signaling pathways to proper vascular growth in humans (21, 23, 27, 32, 40, 69). Aberrant Wnt/β-catenin signaling has also been implicated in most CLDs, highlighting its importance in the study of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) (4–7, 38, 43, 56). To date, Dickkopf-1 (DKK1) is widely accepted as an antagonist of the canonical Wnt signaling pathway as DKK1 inhibits formation of a ternary complex involving LRP5/6, frizzled, and Wnt ligands which results in inhibition of the canonical Wnt signaling (49, 58). It also regulates epithelial proliferation, mesenchymal transition, and migration in a dose-dependent manner through Kremens (Krm) 1 and 2 or cytoskeleton-associated protein 4 (CKAP4) (37, 54). DKK-1 binding of Krm 1 and 2 triggers the internalization and degradation of the LRP receptor (8, 19). Furthermore, DKK1 induces angiogenesis through Wnt/β-catenin-dependent and -independent mechanisms in endothelial and progenitor cells (16, 17, 53, 60). It also regulates lung smooth muscle cell (SMC) differentiation and proliferation (22, 31, 66). To date, the role DKK1 plays in regulating pulmonary microvascular homeostasis remains unknown.

Our previous studies demonstrated that DKK1 expression by MVPCs varies depending on the CLD. DKK1 protein levels are increased in primary MVPCs isolated from patients with pulmonary arterial hypertension (PAH) (both familial PAH and idiopathic PAH), IPF, and COPD (29, 30, 62). In lung tissue corresponding to these CLDs, DKK1 is localized to the endothelial and smooth muscle cell layers of the remodeled vasculature, relative to normal lung tissue. Exposure of primary MVPCs from normal and patients with FPAH induced a profibrotic phenotype with increased expression of α-smooth muscle actin, collagens, and extracellular matrix. Treatment with the DKK1 inhibitor, gallocyanin, decreased the expression of these genes and in some instances reduced the expression to that of the normal MVPCs (29). As DKK1 is a target of β-catenin (50), we recently demonstrated that transgenic mice with stabilized β-catenin and Wnt activation in MVPCs express increased levels of DKK1, which correlated with enhanced adaptive angiogenesis by the microvascular endothelium as well as MVPCs (62). This shows the complex interactions between β-catenin and its upstream inhibitor DKK-1. However, the cell-specific mechanisms by which DKK1 may influence the microvascular niche have not been elucidated.

To address the aforementioned gaps in knowledge, we designed studies to test the hypothesis that DKK1 was a modulator of three cell types comprising a pulmonary microvascular niche, microvascular endothelium (MVECs), MVPCs, and smooth muscle (SMC). In vitro, DKK1 expression differentially affected sprouting and migration of murine and human MVECs and MVPCs as well as the SMC contractile phenotype, correlated to level of Wnt signaling, dose, and CKAP4 receptor expression. In vivo, genetic depletion of lung MVPC DKK1 compared with wild-type (WT) demonstrated tissue stiffening, parenchymal collagen deposition, and increased muscularization. DKK1 KO responded to hypoxia exposure with loss of tissue structure and increased mean linear intercept (MLI). Taken together, the data demonstrate that DKK1 expression is necessary to maintain the microvascular niche, whereas its effects are context specific.

METHODS

Study approval.

The Institutional Animal Care and Use Committee at National Jewish Health approved all animal procedures and protocols. This study used banked patient cell lines obtained using IRB No. 9401 approved by the Vanderbilt University Medical Center IRB Committee, Nashville, TN. Patients were consented under this IRB for the generation and storage of human cell lines. Primary cells were obtained from patients with advanced COPD with severe physiological impairment requiring lung transplantation as previously described (29, 30). Isolated human and murine MVPCs and murine in vivo age-matched models were used. End-point analysis was performed in a blinded fashion. Primary human MVPCs and MVECs were isolated and characterized as previously described (28, 30, 62), or purchased from Lonza (Lonza, Walkersville). Human pulmonary artery smooth muscle cells were purchased from Lonza (Lonza, Walkersville).

Genetic manipulation of murine ABCG2^pos mesenchymal progenitor cells.

ABCG2-Cre^ERT2 mice, a generous gift from Dr. B. Sorrentino (St. Jude Children’s Research Hospital, Memphis, TN) (26), were crossed to a fluorescent enhanced green fluorescent protein (eGFP) reporter (Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP) JAX Stock No. 007676; designated as mT/mG) strain to facilitate lineage analysis and quantitation via eGFP expression. A third gene was crossed into the mice to stabilize β-catenin [Catnb^loxp(Δex3)], a generous gift from Dr. M.M. Taketo (Kyoto University, Kyoto, Japan) (34), designated as βOE. A third gene was crossed into the mice to knockdown DKK1 (Dkk1^tm1.1Svo (55); fl DKK1; MGI:5013423), designated as DKK1 knockdown (KD) (DKK1^+/− or DKK1^−/−).

To knock down MVPCs in vivo, we crossed the Abcg2 driver/reporter strain to a mouse containing a floxp stop allele regulating the expression of diphtheria toxin A (42, 64) (JAX Stock No. 009669). Mice were injected intraperitoneally at 10–12 wk of age with a single low-dose (0.5 mg) tamoxifen (T-5648; Sigma, St. Louis, MO) in sesame oil, or sesame oil alone (vehicle control) (18, 46). The mice were randomized and distributed as 3–5 mice per cage for studies (both male and female). These model systems, as well as isolated MVPCs, were validated as previously described (30, 62) and labeled the cells with the highest expression of Abcg2.

Phenotyping of pulmonary vascular dysfunction and airway physiology.

Pulmonary artery pressure was documented indirectly by the measurement of right ventricular systolic pressure (RVSP) coupled with quasi-static mechanical properties of the lung we measured using the flexiVent invasive plethysmography system (SciREQ, Inc.) as previously described (39). Histologic end points included muscularization and microvessel density of the distal microvessels by immunostaining to detect smooth muscle actin (SMA; Dako clone 1A4) or Factor 8 (A0082 DAKO) on 6–8 mice per group (15). To analyze the changes in distal lung structure, images of hematoxylin-eosin (H&E)-stained lung sections were taken on a Nikon Eclipse 80i microscope using NIS Elements AR software (v 4.13). A minimum of 6–10 nonoverlapping images were taken blinded at ×20 excluding images containing large vessels or airways. Images were then loaded into a macro within Metamorph Software (v 7.5.0.0) for analysis (47). MLI was calculated from the average of all the images.

Quantitation of collagen.

Ten fields of view per two sections, four sections per mouse of trichrome-stained mouse lungs were photographed at ×40 magnification. Resulting color images were scanned to quantitate the number and intensity of blue (collagen) positive pixels relative to red. Images were scanned using Fiji (Image J; v 2.0.0-rc-43/1.51a) using a custom plug-in written by M. Majka (Denver, CO). The tool was written in the ImageJ macro language to detect pixels with a blue intensity over a desired threshold. This macro set all pixels that had the desired blue intensity to the color yellow and outputs a count of the number of yellow pixels. The specified threshold limit was based on a positive and negative control image.

Imaging.

Epifluorescent and bright-field images were captured with Nikon Eclipse 90i upright epifluorescence or Nikon Eclipse TS100 using the Nikon DS-Fi1 camera with NIS elements AR 4.11.00 acquisition software. The BZ-X800 KEYENCE system, with capture and analysis software, was also used. Fluorochromes used included DAPI (to label nuclei), secondary antibodies conjugated to Alexa 488 or Alexa 594 (Thermo Fisher Scientific, Inc., Hampton, NH).

RNA isolation and qPCR.

qRT-PCR was performed in triplicate or with an n of three or greater independent patient samples. Briefly, total RNA was prepared with Qiagen RNA Isolation Kit reagents (Qiagen, Valencia, CA) for total RNA isolation and analysis of gene expression. Complimentary DNA generated from amplified RNA was hybridized to duplicate Affymetrix (Santa Clara, CA) Human Gene 1.0 ST arrays. To determine the cellular response to the DKK1, cells were plated at a concentration of 60,000 cells per well in a medium containing 20% serum. The cells were allowed to remain in 20% serum medium for 24 h. After 24 h, the medium was changed to 20% serum treatment medium containing 50, 100, or 150 ng/mL DKK1 (Origene, Rockville, MD). SMCs were treated with 100 ng/mL of Wnt5A. RNA lysates were collected at 48 h and protein lysates were collected at 72 h for analyses of gene and protein expression. qRT-PCR assays were performed in triplicate (for each of the wells collected) and levels of analyzed genes were normalized to hypoxanthine phosphoribosyltransferase abundance (GAPDH or HPRT) using available TaqMan primer sets.

ECIS.

Human lung MVECs (Lonza, Walkersville) were plated at a concentration of 112,500 cells per well on gelatin-coated 8W1E PET ECIS culture ware arrays (Applied Biophysics, Troy) overnight to achieve confluence. The following day, MVPCs were added at a concentration of 37,500 cells per well. Prior to experiments, the optimal concentration of MVECs and ratio of MVPCs was determined. MVPC lines were normalized by cell number. Controls for these experiments included untreated MVECs and MVECs with wounding. On the third day, the arrays were put on the ECIS Ztheta (Applied Biophysics, Troy, NY). Resistance recordings were performed at 4 kHz every 10 min for 24 h. At 2–3 h, an electrical wound was created by administering a 20-s pulse at 60,000 Hz. In some cases, the pulse was immediately repeated to ensure cell death. In other experiments not including MVPCs, a total 150,000 MVECs were applied per well and allowed to adhere overnight. A concentration of 100 ng/mL of DKK1 was applied to the MVEC monolayer the next day, 6 h before wounding. Recovery from wounding in all experiments was normalized to the first resistance value data collected subsequent to wounding. These experiments were performed with two sample replicates and repeated twice.

Spheroid assays and quantitation.

Confluent WT and βOE MVPCs were passaged for spheroid formation. A micro-mold (MicroTissues Inc. No. 24-906) was used to create a 2% agarose three-dimensional (3-D) petri dish containing 96 individual recesses that allow cells to self-assemble into spheroids. 3-D petri dishes were pretreated with 1 mL of culture medium for 45 min (3 treatments of 15 min each) before cell seeding in a 24-well plate. MVPCs were seeded into the 3-D dish at a density of 500 cells per well in 75 µL total volume per the manufacturer’s recommendation. Cells were allowed to settle for 30 min before adding 1 mL of endothelial medium to the area surrounding the 3-D dish. Cells were allowed to form spheroids for 24 h, at which point the endothelial medium was aspirated and replaced with fresh medium containing either 100 ng/mL DKK1 or no DKK1. Cells were then cultured overnight with or without DKK1. Spheroids were collected by aspirating the medium surrounding the 3-D petri dish and pipetting 500 µL of prewarmed medium into the mold to dislodge spheroids. Spheroid-containing medium was collected into a 15-mL conical tube and centrifuged for 5 min at 110 g. Supernatant was aspirated, and spheroids were resuspended in a solution of 2 mg/mL rat tail collagen (Gibco A10483-01) combined with an equal volume of 0.5% 4,000 cP methylcellulose solution (Sigma M0512) in endothelial medium. Spheroids were then pipetted in 1-mL volumes to a new 24-well plate and incubated for 30 min to polymerize the collagen before adding 500 µL of culture medium with or without DKK1 to each well. After 24 h, spheroids were imaged on a Keyence BZ-X810 digital fluorescent microscope. Keyence BZ-X800 software was used to measure the migration radius of cells from the center of the spheroid.

Statistical analysis.

Data were analyzed by one-way ANOVA followed by Tukey’s honestly significant difference post hoc analysis. Murine qPCR and patient samples were analyzed using nonparametric Wilcoxon/Kruskal–Wallis and a chi-square approximation. All analyses used JMP v 9.0.2. Data presented as ±standard error from the means ± SE. Significance was defined as *P < 0.05, **P < 0.01, or ***P < 0.001.

RESULTS

Vascular localization and altered expression of DKK1 is apparent in a variety of CLD, including PAH, COPD/emphysema, and IPF (29, 30, 62). Although initially described as an inhibitor of canonical Wnt signaling, the functions of DKK1 are concentration and cell type specific (16, 17, 37, 53, 54, 60). Because MVPCs are a central component of the microvascular niche and participate in de novo angiogenesis in response to injury (30), we evaluated the effects of DKK1 on sprouting angiogenesis using a three-dimensional spheroid assay, comparing wild-type (WT) to Wnt-activated (βOE) murine MVPCs. We found that DKK1 administration did not affect sprouting or migration of the WT MVPCs at 24 h (Fig. 1). In contrast, the Wnt-activated βOE MVPCs demonstrated significant increases in sprouting and migration upon treatment (Fig. 1A). DKK1 differentially affected the sprouting and migration of murine MVPCs based on their Wnt/β-catenin signaling status.

Figure 1.

Dickkopf-1 (DKK1) differentially affected mesenchymal vascular progenitor cell (MVPC) numbers and angiogenic sprouting depending on their Wnt signaling status. A and B: wild-type (WT) or Wnt-activated (βOE) MVPC spheroids were plated in collagen and methylcellulose in the absence or presence of DKK1 (100 ng/mL) for 24 h. The radius of sprouts and migrating cells was quantitated. The experiment was repeated twice independently and a total of 21, 21, 23, 24 spheroids was quantitated per group. The cell line was isolated from 10 pooled female mice. Scale bar = 50 μm. C and D: lungs from WT and DKK1 knockdown (KD) MVPC mice were collected 8 wk following low-dose tamoxifen induction and green fluorescent protein (GFP) expression analyzed via lineage tracing (C) or flow cytometry (D). n = 4 or 5; Scale bar = 100 μm. E and F: isolated WT or DKK1 knockdown (DKK1_KD) MVPC spheroids were plated, and the radius of sprouts and migrating cells was quantitated after 48 h. The experiment was repeated twice independently and a total of 11, 11 spheroids was quantitated per group. The cell lines were isolated from five pooled female mice. Scale bar = 50 μm. Data presented as means ± SE. G: cytospins were immunostained to detect expression of DKK1. Scale bar = 50 μm. DAPI, 4′,6-diamidino-2-phenylindole; SMA, smooth muscle actin. *P < 0.05, **P < 0.01, and ***P < 0.001.

To analyze the effects of DKK1 on the microvascular niche in vivo, we genetically depleted the expression of lung MVPC DKK1 via knock down (KD) of one or both alleles (DKK1^{+/− & −/−}) in vivo. DKK1^KD decreased the number of MVPC identified by lineage tracing, confirmed by flow cytometry to detect CD45^neg GFP^pos cells in single-cell suspensions of lung tissue (Fig. 1, C and D). Cell lines were isolated and cultured from each strain as previously described (30, 62). Spheroid analysis of cell sprouting suggested that DKK1^KD cells exhibit decreased migration (Fig. 1, E and F). Decreased expression of DKK1 was confirmed by immunostaining of cytospins from WT or DKK1^KD primary cell lines (Fig. 1G).

We next compared the resulting lung function and structure from mice with activated Wnt signaling (βOE) or DKK1 KD in MVPCs or WT. Both DKK1^KD (DKK1^{+/− & −/−}) and Wnt activation (βOE) in MVPCs resulted in a statistically significant trend of altered lung mechanics characterized by decreased compliance (Crs), increased elastance (Ers), and a downward shift in pressure volume loops (Fig. 2, A–E). Changes in airway physiology were independent of significant alterations to the right ventricular systolic pressure (RVSP) or Fulton’s index (RV/LV + S; Fig. 3, A and B), indicating significant vascular remodeling or the development of pulmonary hypertension (PAH). Subtle alterations in lung mechanics are typically associated with changes in matrix and fibrosis, so we next evaluated the lung parenchyma. Lineage tracing of lung MVPC revealed that they did not express α-smooth muscle actin (α-SMA; Fig. 2, F–H). However, enhanced SMA expression was localized to the interstitium in both βOE and DKK1^KD lung tissue alluding to the presence of myofibroblasts. Trichrome staining to detect collagen deposition and blinded quantitation using Fiji analysis revealed increased collagen in the interstitium of βOE and DKK1^+/− lung tissue relative to WT (Fig. 2, I–O). The increased collagen and tissue mechanics correlated to decreased MLI in the DKK1^KD tissue (Fig. 3J). Interestingly, DKK1^KD lungs also exhibited increased fully muscularized and total microvessels relative to WT and βOE (Fig. 2, P–R).

Figure 2.

Airway function in Dickkopf-1 (DKK1) knockdown (KD) mesenchymal vascular progenitor cell (MVPC) mice demonstrated increased lung stiffening and parenchymal collagen deposition. FlexiVent analysis measurement in wild-type (WT), Wnt-activated (βOE), or DKK1^KD MVPC mice for 8 wk following low-dose tamoxifen induction. FlexiVent analysis of inspiratory capacity (IC) (A), total resistance (Rrs) (B), system compliance (Crs) (C), and elastance (Ers) (D). n = 10, 4, 12 mice per group. E: pressure volume curves and quantitation of the area under the curve. F–H: lineage labeling of WT, Wnt-activated (βOE), or DKK1^KD GFP^pos MVPC in lung tissue. Eight weeks following low-dose tamoxifen induction, mice were euthanized, and lungs were agarose inflated using constant pressure, to obtain lung tissue for histological and immunofluorescent analyses. Scale bar = 100 μm. α-Smooth muscle actin (α-SMA) was also localized by immunodetection. n = 5, 4, 4. I–N: representative bright-field images of trichrome-stained lung tissue (L–N enlarged panels); collagen deposition is visible in blue. O: quantitation of parenchymal collagen via Fiji/ImageJ v2.0 2018. n = 5, 4, 4 mice per group. Immunostaining was performed on lung tissue sections to detect α-smooth muscle actin (α-SMA) and Factor 8 (F8) positive microvessels ranging from 0 to 50 μm in diameter. Scale bar = 100 μm. P: degree of muscularization was analyzed as <50% SMA positive being partial and >50% SMA positive, fully muscularized. Q: total numbers of SMA-positive microvessels. R: F8 positive microvessels per field of view (f.o.v.). Immune-positive microvessels were counted in 20 f.o.v. per mouse at ×20 magnification. Data presented as means ± SE. n = 5, 4, 4 mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3.

Hypoxia exposure drives loss of lung structure in Dickkopf-1 (DKK1) knockdown (KD) mesenchymal vascular progenitor cell (MVPC) mice. Two weeks following low-dose tamoxifen induction, WT, Wnt-activated (βOE), or DKK1^KD mice were randomized and exposed to relative hypoxia (10% oxygen) or room air (RA) at Denver altitude for 6 wk to induce pulmonary hypertension (PAH). At 8 wk, lungs were agarose inflated using constant pressure, to obtain lung tissue for histological analyses. Right ventricular systolic pressure (RVSP) and FlexiVent analyses were performed. Indices of PAH were analyzed including Fulton’s index (RV/LV + S) (A) and RVSP (B) measured by a pressure transducer placed in the right ventricle. C: hematocrit increased with hypoxia. Data presented as means ± SE. The mean is indicated by +. n = 10, 4, 12, 9, 5, 14 mice/group. FlexiVent analysis of inspiratory capacity (IC) (D), total resistance (Rrs) (E), system compliance (Crs) (F), and elastance (Ers) (G). H: pressure volume curves and quantitation of the area under the curve. Loss of tissue structure was quantitated by analysis of mean linear intercept (MLI) (I). Data presented as means ± SE. n = 9,5,14 mice per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Congruous to these findings, we have previously reported that Wnt-activated βOE MVPCs express increased DKK1, whereas lungs exhibited decreased muscularization, SMA expression as well as contractility (30, 62). Therefore, we next evaluated the effect of DKK1 depletion in a murine model of hypoxia-induced pulmonary hypertension (PAH), which is characterized by changes in microvascular structure and function. The mouse strains all responded to hypoxia exposure (10% Denver altitude) with increased Fulton’s index, RVSP, and hematocrit (Fig. 3, A–C). In terms of airway physiology, the most significant changes were in the hypoxia-exposed DKK1^KD mice, relative to the room air (RA) DKK1^KD mice (Figs. 2, A–D, and 3, D–G). The hypoxia-exposed DKK1^KD mice exhibited increased inspiratory capacity (P < 0.0001), Crs (P < 0.0002), Ers (P < 0.0013), and a downward shift in pressure volume loops relative to RA (Fig. 3H). This relative “normalization” in function and levels approaching WT were related to a loss of distal lung tissue structure indicated by increased mean linear intercept (MLI; Fig. 3I).

Our data suggest that MVPC expression of DKK1 influences the microvasculature as well as tissue structure. Therefore, it is reasonable to speculate that the function of MVPC is regulated by the disease microenvironment, to influence the microvasculature. DKK1 expression by MVPCs varies depending on the CLD (29, 30). DKK1 transcript expression is increased in Wnt-activated murine MVPCs, which correlates with increased levels in COPD MVPCs (62). DKK1 is known to have varying effects on Wnt activation as well as angiogenesis, in a concentration- and receptor-dependent fashion (17, 19, 53). Thus, we sought to evaluate the effects of DKK1 on the angiogenic phenotypes of normal compared with COPD lung MVECs, COPD lung MVPCs as well as normal lung vascular SMCs (Figs. 4 and 5).

Figure 4.

Effect of Dickkopf-1 (DKK1) on the recovery of endothelial barrier function following injury is dependent on CKAP4. The effects of exogenous DKK1 on human lung microvascular endothelium cells (MVECs) were analyzed. A: human lung MVECs, normal and chronic obstructive pulmonary disease (COPD), were treated with 0 or 100 ng/mL DKK1. qPCR analysis was performed to compare selected differences in gene expression. Selected genes included the DKK1 receptor, CKAP4, the WNT5A receptor, ROR2, Wnt target, AXIN2, the cytoskeletal elements ACTA2, TAGLN, and the migratory receptor ligand complex, ROBO2 and SLIT2. Analysis was performed in triplicate for each sample. *Difference from vehicle control. B–E: The electrical wounding injury on recovery of barrier function of normal or COPD MVEC (with low or high expression of CKAP4) in the absence or presence of DKK1 (0 or 100 ng/mL) was measured by ECIS. Resistance was measured (ohms) over time. Groups were color coded and are indicated in the legend. Quantitation of normalized resistance at indicated time points (Δ) was presented in bar graph format. B: three-time points indicated by Δ presented in bar graph format. *Difference from normal unless indicated by a line. C–E: one-time point indicated by Δ presented in bar graph format. Analysis was performed in duplicate or triplicate for each sample and repeated twice independently. Data presented as the means ± SE. *Difference from vehicle control. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5.

Dickkopf-1 (DKK1) regulates the phenotype of mesenchymal vascular progenitor cells (MVPC) and smooth muscle cell (SMCs) in a cell- and dose-dependent manner. The effects of exogenous DKK1 on human lung MVPC and SMC were analyzed. qPCR analysis was performed to compare selected differences in gene expression. Selected genes included the DKK1 receptor, CKAP4, the WNT5A receptor, ROR2, Wnt targets, AXIN2 and PROX1, and the cytoskeletal elements ACTA2, TAGLN. A: human lung MVPCs, normal and chronic obstructive pulmonary disease (COPD), were treated with 0 or 50 ng/mL DKK1. *Difference from vehicle control. B: pulmonary vascular SMCs were treated with 0 or 50 ng/mL DKK1. Analysis was performed in triplicate for each sample. Data presented as means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001.

We first examined the expression of DKK1 receptors by normal and COPD MVECs. Individual MVEC isolates from COPD lung explants varied in their expression of CKAP4 (Fig. 4A), facilitating the comparison between lines with low or high levels of receptor expression. In addition to CKAP4 expression, there were additional baseline differences between the CKAP4^low and CKAP4^high COPD MVEC lines, including their expression of AXIN2, ACTA2, TAGLN, and ROR2. We next tested the effect of DKK1 on primary human lung MVEC phenotype and function based on their expression of CKAP4. The CKAP4^low and CKAP4^high COPD MVECs responded to DKK1 with opposite trends in the expression of ACTA2, TAGLN, and SLIT2 (Fig. 4A). In the CKAP4^high COPD MVECs, DKK1 treatment decreased both CKAP4 and ROR2 receptor expression. The ability of MVECs to form a functional barrier was next evaluated following electrical wounding, in the absence or presence of DKK1 (Fig. 4, B–E). Following wounding, normal and CKAP4^low MVECs recovered their barrier function at a faster rate than the CKAP4^high COPD MVECs (Fig. 4B). However, both the CKAP4^low and CKAP4^high COPD MVECs plateaued at a much lower resistance than MVECs from healthy donors, suggesting that COPD MVECs either migrate more slowly or do not form a stable barrier. The presence of DKK1 decreased the rate at which barrier function was regained following injury in normal and CKAP4^high COPD MVECs (Fig. 4, C and E). DKK1 had no measurable effect on the CKAP4^low COPD MVECs (Fig. 4D). Since DKK1 had no effect on MVEC proliferation (not shown), these data suggest DKK1 impacted the MVEC migration and barrier formation. Thus, elevated levels of DKK1 in COPD and emphysema may decrease vascular integrity, both important in angiogenesis as well as in overall tissue repair.

Similar to MVECs, DKK1 affected MVPCs in a dose-dependent and cell-specific manner. Exogenous DKK1 had no effect on the canonical Wnt targets AXIN2 and PROX1 in MVPCs from healthy individuals; however, it increased the expression of both targets in COPD MVPCs at the lower dose (Fig. 5A). Although counterintuitive, we hypothesized that this effect was likely receptor-dependent. Indeed, we found that COPD MVPCs had significantly increased levels of CKAP4 and ROR2, the receptors for DKK1 and WNT5A, respectively (11, 37, 48). The expression of both receptors, however, was downregulated by DKK1 (Fig. 5A).

Since DKK1 and Wnt signaling play a major role in vSMC developmental specification, and since MVPCs likely impact vascular SMC remodeling function in the microvascular niche, we next defined the effect of DKK1 on isolated human lung SMC (22). DKK1 did not increase the canonical Wnt targets AXIN2 and PROX1 (Fig. 5B). SMC expressed both CKAP4 and ROR2 and responded to DKK1 with significant decreases in the contractile cytoskeletal transcripts ACTA2 and TAGLN (Fig. 5B). These data further support findings in our murine model in which Wnt activation in MVPCs resulted in a loss of microvascular-associated smooth muscle αactin expression. In addition, we analyzed the effect of WNT5A on SMC phenotype because, first, DKK1 and WNT5A reciprocally regulate each other’s activity; second, both COPD MVPC and murine Wnt-activated MVPCs express increased levels of WNT5A (62), and finally, COPD MVPCs expressed increased level of the WNT5A receptor, ROR2 (Fig. 5A). Similar to DKK1, WNT5A decreased the expression of the cytoskeletal proteins ACTA2 and TAGLN while not affecting AXIN2 (Fig. 5B). Taken together, these data illustrate cell-specific effects of the MVPCs production of the Wnt modulator DKK1 within the microvascular niche (Fig. 6).

Figure 6.

Dickkopf-1 (DKK1) regulates the individual functions of and paracrine effects between cells of the microvascular niche, including mesenchymal vascular progenitor cell (MVPCs), microvascular endothelium cells (MVEC) and smooth muscle cells (SMC). DKK1 regulates MVPC function depending on their Wnt/β-catenin activation status. MVPC production of DKK1 regulates lung tissue function, structure, and parenchymal collagen deposition, which correlate to DKK1 dependent effects on microvascular muscularization and density. In vitro, DKK1 regulates MVEC barrier function and in migration likely through a CKAP4-dependent mechanism. It also regulates Wnt/β-catenin signaling by MVPC in a dose-dependent manner. Finally, DKK1 decreases the expression of contractile proteins by vascular SMC, similar to Wnt5A.

DISCUSSION

The differential expression of DKK1 by human and murine Wnt-activated MVPCs suggested a potential mechanism for paracrine regulation of the microvascular niche during the development of CLDs (28–30, 62). In this study, we show that depletion of DKK1 in Abcg2^pos lung mouse adult tissue resident vascular precursors (MVPC) alters lung mechanics, parenchymal collagen deposition as well as of microvessel muscularization and density. To complement the in vivo mouse modeling, we have identified cell- or disease-specific responses to DKK1, in primary COPD MVPCs as well as COPD MVECs, supporting a paradoxical disease-specific response of cells to well-characterized factors. DKK1 also regulated the expression of contractile determinants by lung vascular SMCs. Responses to DKK1 were dose-dependent and correlated with varying expressions of the DKK1 receptor, CKAP4.

Although DKK1 is known to regulate lung branching morphogenesis (17, 22, 31), our results suggest that DKK1 exerts effects on the angiogenic potential of lung MVPCs depending on their level of active canonical Wnt signaling. Wnt-activated MVPCs respond to DKK1 stimulation with enhanced sprouting and migration, relative to wild-type MVPCs. Our previous work also demonstrated that primary lung MVPCs from normal versus patients with Wnt-activated pulmonary hypertension respond differently to DKK1, with the diseased cells expressing increased levels of collagen and fibronectin as well as smooth muscle αactin (29). Thus, the proangiogenic effects of DKK1 with activated Wnt signaling may be dependent on MVPC expression of Wnt target genes, matrix, or integrins.

Selective depletion of DKK1 expression by MVPC in adult mice resulted in decreased lung function indicative of altered lung mechanics in the absence of detectable changes in cardiovascular physiology. The detrimental changes in physiology were correlated to increased appearance of myofibroblasts and parenchymal collagen in the lung tissue as well as decreased MLI and number of MVPC. Previous studies also link decreased MVPC with increased MLI and the development of emphysema (62). Interestingly, Wnt activation in MVPC yielded a similar effect on the lungs, to a lesser degree. In the presence of vascular injury induced by the exposure of the strains to hypoxia-driven PAH, loss of tissue structure results in the DKK1^+/− strain. Both results indicate the significance of spaciotemporal regulation of Wnt signaling and DKK1 expression by MVPCs. Our current studies are directed toward understanding how DKK1-regulated parenchymal mechanics may result in loss of tissue structure and whether DKK1 or receptor distribution determines whether fibrosis or emphysema develops. We speculate that regional changes in DKK1 or CKAP4 expression could explain why one patient develops both fibrosis and emphysema in combined pulmonary fibrosis and emphysema (CPFE), which is distinct from fibrosis or emphysema alone (3, 44).

The activation, migration, or proliferation of myofibroblasts as well as subsequent collagen deposition is attenuated by DKK1 expression likely via reciprocal inhibition of canonical Wnt signaling (1, 56). DKK1 also regulates the profibrogenic transforming growth factor-β (TGF-β), platelet derived growth factor (PDGF), and connective tissue growth factor (CTGF) signaling pathways in myofibroblasts and vascular cells by inhibiting activated MAPK and JNK-signaling cascades (56). In addition, TGF-β stimulates canonical Wnt signaling in a p38-dependent manner by decreasing the expression of the Wnt antagonist Dickkopf-1, conversely, increased DKK1 expression attenuates TGF-driven fibrosis (1). Increased expression of DKK1 with subsequent inhibition of Wnt signaling following treatment may be used as a prognostic marker of improvement in systemic sclerosis (20, 67). Thus, it is not surprising that differential expression of DKK1 has been demonstrated in tissue from patients with IPF and systemic sclerosis (1, 54).

An additional characteristic of CLDs is abnormal microvascular remodeling and angiogenesis (41, 65). We found that with decreased MVPC DKK1 expression, there was both increased microvessel muscularization and density, absent in the other groups. DKK1 appears to direct lung vSMC homeostasis by regulating smooth muscle α actin expression (22, 31, 66). Converse to the in vivo result, in vitro, exogenous DKK1 also decreased the expression of contractile proteins in vSMC. These data also support the hypothesis that the increased levels of DKK1 produced by both βOE MVPCs and COPD MVPCs decrease the SMA^POS vSMC in Wnt-activated MVPC lungs (30, 62), influencing SMC phenotype as well as function. During microvessel regression, vascular SMC may also express lower levels of SMA, exhibiting loss of the contractile phenotype as opposed to apoptosis (45). Taken together, these results support that MVPC production of DKK1 likely influences vascular SMC phenotype and function.

The role for DKK1 in angiogenesis is well supported (16, 17, 53, 60), however, the mechanisms are not well defined. These studies show a cell-specific effect of DKK1 on primary human lung MVECs from normal and patients with COPD. The cells expressed heterogeneous levels of the DKK1 receptor, CKAP4 and exhibited significant differences in the ability to migrate and repair barrier function. Decreased barrier repair in the presence of DKK1 correlated to increased expression of CKAP4. Previous studies have linked DKK1-dependent endothelial cell migration in collagen to the expression of β1 integrin and level of canonical Wnt signaling (9). More recently, CKAP4 was shown to interact with β1 integrin and coordinate integrin recycling and subsequent cell adhesion and migration (52). COPD MVPCs express increased levels of CKAP4 as well as the Wnt5A receptor, ROR2. They respond to DKK1 in a concentration-specific manner, at low concentrations Wnt signaling is enhanced. Together, these data suggest a role for Wnt-DKK1-CKAP4 signaling in microvascular angiostasis as well as angiogenesis.

In conclusion, our results provide new insight into MVPCs expression of DKK1 in the microvascular niche during lung homeostasis. Ongoing studies in our laboratory are designed to better understand the complexity of this niche and interactions at a single-cell level, DKK1 regulation of the inflammatory repertoire in lung as well as impacts on epithelial cells in the distal lung. Because CKAP4 was differentially expressed in COPD MVPCs versus normal as well as heterogeneously in COPD MVECs, it appeared a reasonable regulatory candidate to further evaluate to understand the role of Wnt and DKK1 signaling in the microvascular niche during tissue homeostasis, adaptive angiogenesis, and during the development of CLDs.

GRANTS

This work was funded by NIH grant R01HL116597 (to S.M. Majka) and NIH grant R01HL136449 (to S. M. Majka). Additional funding includes: VA 5 IK2 BX 003841-02 (to B.W. Richmond), FAMRI CIA160005 (to P. Geraghty), and the Alpha-1 Foundation 614218.

DISCLOSURES

Although not directly related to this work, J. Kropski reports advisory board fees for Boehringer Ingleheim, study support from Genentech, and grants from Boehringer Ingelheim. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

M.E.S., B.W.R., M.G., and S.M.M. conceived and designed research; M.E.S., B.W.R., J.A.K., S.A.M., and S.M.M. performed experiments; M.E.S., B.W.R., S.A.M., J.A.B., J.B., P.G., and S.M.M. analyzed data; M.E.S., B.W.R., J.A.B., A.K.H., J.B., M.G., I.P., R.F.F., P.G., and S.M.M. interpreted results of experiments; M.E.S., P.G., and S.M.M. prepared figures; M.E.S., P.G., and S.M.M. drafted manuscript; M.E.S., B.W.R., J.A.K., J.A.B., A.K.H., I.P., R.F.F., P.G., and S.M.M. edited and revised manuscript; M.E.S., B.W.R., J.A.K., S.A.M., J.A.B., A.K.H., J.B., M.G., I.P., R.F.F., P.G., and S.M.M. approved final version of manuscript.