Pregnant rat dams (200–250-gram body weight) were exposed to filtered air (Control), vehicle [Blu, aerosolized propylene glycol/vegetable glycerin (PG/VG) with gold leaf flavoring, https://us.blu.com], or e-cig (Blu, aerosolized PG/VG with 2.4% nicotine with gold leaf flavoring) through a previously established e-cig delivery system from embryonic day 6 (E6) until postnatal day 21 (PND21) [33,34,35,36]. Mimicking real-life puffing topography, the exposure protocol included 4 second puff exposure, 26 second interval between puffs, 6 puffs per vaping episode, 2 vaping episodes per hour, 12 vaping hours per day. All dams were given free access to food and water and were maintained in a 12hr:12hr light:dark cycle. Vaping exposure was skipped on the dams’ delivery date and was resumed 24 hours later. After spontaneous delivery at term, some pups were sacrificed the next day for blood collection. PND1 pups were placed on heating pads to facilitate blood circulation, 3 millimeter Goldenrod animal lancets (Medipoint, Inc.) were used to puncture the submandibular vein and blood was collected for serum cotinine level measurement. Pups were then anesthetized in an isoflurane chamber and dissected to collect tissues for other purposes. The remaining pups were breast fed ad libitum until PND21, when they were sacrificed for BMSC isolation and culture following previous methods, described briefly below [24,37,38]. The experiments (control, vehicle, and e-cig nicotine exposure) were conducted four times. BMSCs were isolated from pooled marrows of 3–4 offspring from each group at postnatal day 21. All animal procedures were performed following the guidelines of the National Institutes of Health for the care and use of laboratory animals after approval of the Lundquist Institute Animal Care and Use Committee (protocol # 31355–03).

As noted above, at PND1, pups were sacrificed, and blood was collected and then centrifuged at 2,500 × g at 4°C for 10 minutes to obtain serum. A 20 μL of serum was processed with 80 μL of methanol containing internal standard, vortex mixed, filtered, and centrifuged at 1500×g at room temperature for 5 min. Serum cotinine levels were measured using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS).

The medullary cavities of rat femurs were flushed with Minimal Essential Medium (MEM) Alpha (1×) + GlutaMax^™−1 (Life Technologies, Catalog# 32561–037) containing 1% penicillin-streptomycin. The cells were washed once with MEM and plated at 1 × 10⁶ cells per T75 flask (Corning, Catalog # 430641U) in the complete medium: MEM Alpha containing 10% fetal bovine serum (FBS) and 1% antibiotic and antimycotic (Gibco, catalog# 15240–062), and cultured at 37°C in 5% CO₂. Non-adherent cells were removed, and fresh media was added every 48 h. At confluence, the cells were harvested and using magnetic beads (Miltenyi Biotech, Auburn, CA), macrophages were depleted with anti-CD11b antibody, and other hematopoietic cells were removed using an anti-CD45 (both from BD Biosciences, Palo Alto, CA). Cells were collected in 1 ml of DMEM supplemented with 10% FBS, and then cultured and passaged. Due to >95% purity of cells at passage (P) 3, all experiments were conducted at P3.

Protein extraction and immunoblot analysis for proliferating cell nuclear antigen (PCNA, a homotrimer, which by encircling the DNA acts as a scaffold to recruit proteins involved in DNA replication), β-catenin, lymphoid enhancer binding factor 1 (LEF-1), peroxisome proliferator-activated receptor gamma (PPARγ), adipose differentiation-related protein (ADRP), CCAAT-enhancer-binding protein alpha (C/EBPα), α-smooth muscle actin (αSMA), calponin, and fibronectin were performed as previously described [39,40,41]. Briefly, cells were homogenized in 10 mM Tris (pH 7.5), 0.25 M sucrose, 1 mM EDTA, 5 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, and 10 μg/ml each of pepstatin A, aprotinin, and leupeptin, and centrifuged at 16000×g for 10 min at 4°C. Equal amounts of protein from the supernatant were dissolved in electrophoresis sample buffer, loaded to SDS polyacrylamide (4–12% gradient) gel and separated, followed by electrophoretic transfer to a nitrocellulose membrane. The membrane was stained with 0.1% Ponceau S in 5% acetic acid (Sigma-Aldrich, Catalog# P-7170) and then cut into several strips according to each marker’s molecular weight. The membrane strips were blocked with 5% non-fat dry milk in 1× Tris-buffered saline containing 0.1% Tween 20 for 1 h, and then incubated with the corresponding primary antibody from Proteintech Group Inc. including fibronectin (1:1000, Catalog# 15613–1-AP), LEF-1 (1:500, Catalog# 14972–1-AP), PPARγ (1:500, Catalog# 16643–1-AP), ADRP (1:800, Catalog# 15294–1-AP)), and C/EBPα (1:800, Catalog# 18311–1-AP) except αSMA (1:20,000, Sigma-Aldrich, Catalog# 2547), Calponin (1:8,000, Sigma-Aldrich, Catalog# C2687), β-Catenin (1:800, Santa Cruz, Catalog# sc-7963), PCNA (1:2,000, NOVUS, Catalog# NB500–106), and β-actin (1:2,000, Cell Signaling, Catalog# 13E5) overnight at 4°C. Subsequently, the membrane strips were washed with 1× Tris-buffered saline + 0.1% Tween 20 and incubated with the appropriate secondary antibody for 1 h at room temperature, washed again, and developed with SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology, Rockford IL) following the manufacturer’s protocol. Membrane strips corresponding to the molecular weight of β-actin were stripped with Restore^™ Western Blot Stripping Buffer (Pierce, Catalog# 21059), blocked and re-probed with the appropriate primary and secondary antibodies. The densities of the PCNA, β-catenin, LEF-1, PPARγ, ADRP, C/EBPα, αSMA, calponin, and fibronectin bands were quantitated using ImageJ software (National Institutes of Health) and normalized to β-actin.

Cell proliferation was assessed via 1) colorimetric tetrazolium-formazan assay using CellTiter 96^® Aqueous One Solution Cell Proliferation Assay (Promega, Catalog #G3580) and 2) Western blot analysis for PCNA. For tetrazolium-formazan assay, BMSCs were plated at 5 × 10⁴ cells per well and 100 μl complete medium: MEM Alpha containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (Gibco, Life Technologies, Grand Island, NY, catalog# 15240–062) and cultured at 37°C in 5% CO₂ for 72 hours, with fresh media added every 24 hours. Following 72 hours, 20 μl/well of Cell Titer 96 Aqueous One Solution Reagent was added, and plate was incubated at 37°C in 5% CO₂ for 3 hours. Absorbance at 490 nm was measured using a 96-well plate reader. Western blotting for PCNA was performed as outlined above.

To assess cell migration, Boyden transwell cell migration assay using 24-Well Colorimetric Cell Migration Assay (Millipore, Catalog # ECM 508) was used. BMSCs were suspended at 5 × 10⁵ cells/well in 10% FBS, 300 μl cell suspension was added to each insert, and 500 μl serum free media (MEM w/Glutamax + 5% BSA) was added to the lower/outer chamber. Plate was incubated for 24 hours at 37°C in 5% CO₂. 400 μl Cell Stain provided by the kit was added, and non-migratory cells were removed. Extraction buffer was added, and after 15 minutes, optical density was measured at 560 nm. The cell migration assay was performed under both normoxia (21% O₂) and hyperoxia (95% O₂). Cells were exposed to hyperoxia in Billups-Rothenberg chambers (Del Mar, CA) as described previously [42,43].

To assess wound healing, the wound healing assay kit (Abcam, Catalog # ab242285) was used. Briefly, ensuring the alignment of wound fields and firm contact of inserts to the bottom of the plate wells, specific volumes of cell suspension according to cell concentration [5 × 10⁵ cells/ml in medium containing 10% fetal bovine serum (FBS)] were added to each well. For optimal cell dispersion, half of the specific volumes of cell suspension were added to either side of the open ends at the top of the insert. After incubating for 48 hours, inserts were slowly removed from all wells using sterile forceps. For wound field baseline cells, wells were washed 3 times with PBS containing calcium and magnesium, stained with 400 μl of cell stain solution provided by the manufacturer and allowed to incubate for 15 mins at room temperature. Cell stain was aspirated, wells washed carefully 3 times using deionized water and allowed to dry at room temperature before imaging. For wound healing cells, media were slowly aspirated, and wells were washed with media to remove dead cells and debris before adding fresh media with FBS for continued culture. Closure was monitored under a light microscope. 24 hours after inserts were removed, wells were washed, stained, and imaged following the protocol for wound field baseline cells.

The data were analyzed, using either one way ANOVA with Tukey’s post-hoc analysis for multiple comparisons or student’s t-test, as appropriate. The results are based on four independent experiments and the values are expressed as mean ± SE. A P value of <0.05 is considered to represent statistically significant difference between the experimental groups.

Compared to Fresh air (FA) controls, serum cotinine levels were significantly increased in e-cig with nicotine exposed group (Fig. 1).

Cell proliferation, as determined by tetrazolium-formazan assay and Western blotting for PCNA (proliferating cell nuclear antigen), a key nucleic marker that plays an essential role in nucleic acid metabolism as a component of cell replication and repair machinery, was significantly inhibited in e-cig exposed cells compared to controls (p<0.01), while there was no significant difference in cell proliferation between the control and vehicle exposed BMSCs (Fig. 2).

In normoxia, BMSCs from control and vehicle exposed cells showed similar migration, while BMSCs from e-cig exposed cells demonstrated only 50% migration vs. control and vehicle exposed cells (p < 0.01). In hyperoxia, BMSCs from all groups demonstrated higher migration vs. the corresponding normoxia groups; however, even in hyperoxia, BMSCs from the e-cig exposed group demonstrated markedly reduced migration vs. the corresponding control and vehicle exposed groups which showed similar migrations (Fig. 3).

Complementing the BMSC proliferation and migration data, at 24h time point, BMSCs from control and vehicle exposed group demonstrated similar, i.e., 94–95% wound closure, while e-cig exposed BMSCs exhibited significantly reduced (43%) wound closure (Fig. 4).

The protein level of PPARγ (Fig. 5a), which is a key nuclear transcription factor that determines lipofibroblastic phenotype, was significantly inhibited in nicotine-containing e-cig BMSC exposed group versus control and vehicle exposed groups which exhibited statistically similar levels. Similar effects were seen with ADRP (Fig. 5b) and C/EBPα (Fig. 5c) protein levels, two known downstream targets of PPARγ. In contrast, the protein levels of myogenic markers fibronectin (Fig. 6a), αSMA (Fig. 6b) and calponin (Fig. 6c) were significantly higher in BMSCs from nicotine-containing e-cig exposure group vs. control and vehicle exposed groups, which exhibited similar protein levels of these myogenic markers.

Having determined upregulation of myogenic proteins in e-cig exposed BMSCs, the protein expression of key intermediates (β-catenin and LEF-1) of Wnt signaling, which is the main determinant of cellular myofibroblastic phenotype, was determined. Compared to control and vehicle exposed groups, protein levels of β-catenin (Fig. 7a) and LEF-1 (Fig. 7b) were significantly increased in the e-cig exposed BMSCs, but there were no significant differences between the control and vehicle exposed groups.