Effect of Perinatal Vitamin D Deficiency on Lung Mesenchymal Stem Cell Differentiation and Injury Repair Potential - PubMed (original) (raw)

Effect of Perinatal Vitamin D Deficiency on Lung Mesenchymal Stem Cell Differentiation and Injury Repair Potential

Reiko Sakurai et al. Am J Respir Cell Mol Biol. 2021 Nov.

Abstract

Stem cells, including the resident lung mesenchymal stem cells (LMSCs), are critically important for injury repair. Compelling evidence links perinatal vitamin D (VD) deficiency to reactive airway disease; however, the effects of perinatal VD deficiency on LMSC function is unknown. We tested the hypothesis that perinatal VD deficiency alters LMSC proliferation, differentiation, and function, leading to an enhanced myogenic phenotype. We also determined whether LMSCs' effects on alveolar type II (ATII) cell function are paracrine. Using an established rat model of perinatal VD deficiency, we studied the effects of four dietary regimens (0, 250, 500, or 1,000 IU/kg cholecalciferol-supplemented groups). At Postnatal Day 21, LMSCs were isolated, and cell proliferation and differentiation (under basal and adipogenic induction conditions) were determined. LMSC paracrine effects on ATII cell proliferation and differentiation were determined by culturing ATII cells in LMSC-conditioned media from different experimental groups. Using flow cytometry, >95% of cells were CD45-ve, >90% were CD90 + ve, >58% were CD105 + ve, and >64% were Stro-1 + ve, indicating their stem cell phenotype. Compared with the VD-supplemented groups, LMSCs from the VD-deficient group demonstrated suppressed PPARγ, but enhanced Wnt signaling, under basal and adipogenic induction conditions. LMSCs from 250 VD- and 500 VD-supplemented groups effectively blocked the effects of perinatal VD deficiency. LMSC-conditioned media from the VD-deficient group inhibited ATII cell proliferation and differentiation compared with those from the 250 VD- and 500 VD-supplemented groups. These data support the concept that perinatal VD deficiency alters LMSC proliferation and differentiation, potentially contributing to increased respiratory morbidity seen in children born to mothers with VD deficiency.

Keywords: asthma; lung development; lung regeneration; paracrine effect; reactive airway disease.

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Figures

Figure 1.

Figure 1.

Lung mesenchymal stromal cells exhibit stem cell characteristics based on the presence or absence of specific cell surface markers. (A) FSC and SSC gating strategy to isolate live cells; the percentage of isolated cells (82.9%) is shown. (B) Analysis with flow cytometry–identified cell population from various vitamin D-supplemented groups shows cells to be predominantly CD45-ve, CD90 + ve, CD105 + ve, and Stro-1 + ve (N = 4). FSC = forward scatter; SSC = side scatter; VD = vitamin D.

Figure 2.

Figure 2.

Perinatal VD deficiency decreases lung mesenchymal stem cell (LMSC) proliferation. (A and B) By both the tetrazolium dye assay and IB for PCNA (proliferating cell nuclear antigen), VD-deficient and 1,000 VD–supplemented group LMSCs demonstrated significantly decreased proliferation compared with the 500 VD–supplemented group. Values are means ± SEM; N = 4 (with three replicates/sample). *P < 0.05 (VD-deficient and 1,000 VD–supplemented groups vs. 500 VD–supplemented group). IU = international unit.

Figure 3.

Figure 3.

Perinatal VD supplementation impacts LMSC adipocyte differentiation in a dose-dependent manner. (A) Cells from the VD-deficient and 1,000 VD–supplemented groups demonstrated a significantly reduced uptake of the radiolabeled triglyceride [3H]triolein compared with the 250 VD– and 500 VD–supplemented groups. Values (expressed as cpm normalized to mg of protein) are means ± SEM; N = 3 (with three biological replicates/group). *P < 0.05 (VD-deficient and 1,000 VD–supplemented groups vs. 250 VD– and 500 VD–supplemented groups). (B) The protein concentrations of lipogenic markers PPARγ (peroxisome proliferator-activated receptor γ) and ADRP (adipocyte differentiation-related protein), normalized to GAPDH, decreased significantly in the VD-deficient and 1,000 VD–supplemented groups. Values are means ± SEM; N = 3/group. *P < 0.05 (VD-deficient, 500 VD–supplemented, and 1,000 VD–supplemented groups vs. 250 VD–supplemented group). (C) Cells from the 250 VD– and 500 VD–supplemented groups clearly showed marked Oil Red O staining (N = 3; representative images shown). Arrows indicate increased presence of ORO staining when compared to other conditions. (D) VDR (VD receptor) expression by cells isolated from various experimental groups showed a dose-dependent effect by Western analysis. The highest VDR expression was noted in the 0 VD group and lowest in the 1,000 VD–supplemented group. 0 VD group = VD-deficient group; cpm = counts per minute; ORO = Oil Red O.

Figure 4.

Figure 4.

Perinatal VD deficiency upregulates Wnt signaling and causes myogenic differentiation of LMSCs. (A) Concentrations of LEF-1 (lymphoid enhancer–binding factor 1), a key Wnt signaling intermediate, and fibronectin, a key myogenic protein, normalized to GAPDH, were highest in the lungs of the VD-deficient animals, and the concentrations of these proteins were significantly inhibited in the 250 VD–, 500 VD–, and 1,000 VD–supplemented groups. Values are means ± SEM; N = 3 for each group. *P < 0.05 (vs. VD-deficient group); note that a common GAPDH blot is shown for ADRP (Figure 3B) and fibronectin (A). (B) Immunofluorescence staining showed a decrease in the adipogenic protein PPARγ (red) and an increase in the myogenic protein α-SMA (green) in the VD-deficient group versus the 250 VD–supplemented group. The accompanying histograms show means ± SEM fluorescence intensity (N = 3). α-SMA = α-smooth muscle actin.

Figure 5.

Figure 5.

In vitro VD supplementation blocks TGF-β (transforming growth factor β)–induced myogenic differentiation of LMSCs isolated from the perinatal VD-deficient group. LMSCs isolated from various VD-supplemented groups were exposed in vitro to TGF-β, whereas LMSCs from the VD-deficient group were also pretreated with VD (1 × 10−9 M or 1 × 10−7 M) before TGF-β exposure. TGF-β exposure to LMSCs derived from the VD-deficient group resulted in significantly higher fibronectin but lower PPARγ protein concentrations compared with the concentrations of these proteins in LMSCs derived from the 250 VD– and 500 VD–supplemented groups. LMSCs derived from the 1,000 VD–supplemented group responded similarly to the cells derived from the VD-deficient group. VD pretreatment of LMSCs isolated from the VD-deficient group blocked TGF-β–induced increase in fibronectin (with both doses of VD examined) and decrease in PPARγ concentration (with 1 × 10−7 M dose VD). Values are means ± SEM; N = 3 for each group. #P < 0.05. *P < 0.05 and **P < 0.01; note that a common GAPDH blot is shown for fibronectin and PPARγ.

Figure 6.

Figure 6.

In vitro VD supplementation blocks TGF-β–induced Wnt activation of LMSCs isolated from the perinatal VD-deficient group. (A) LMSCs isolated from various VD-supplemented groups were exposed in vitro to TGF-β, whereas LMSCs from the VD-deficient group were also pretreated with VD (1 × 10−9 M or 1 × 10−7 M) before TGF-β exposure. In vitro VD pretreatment (both doses examined) blocked TGF-β–induced increase in β-catenin and LEF-1 protein concentrations in LMSCs isolated from the perinatally VD-deficient group. LMSCs isolated from perinatally 250 VD–supplemented group also exhibited blockage of TGF-β–mediated increases in β-catenin and LEF-1 protein concentrations. Values are means ± SEM; N = 3 for each group. #P < 0.05 and *P < 0.05; note that a common GAPDH blot is shown for β-catenin and LEF-1. (B) LMSCs isolated from the VD-deficient group were treated with TGF-β with and without pretreatment with VD (1 × 10−7 M). Despite the already higher basal myogenic protein concentrations seen in LMSCs isolated from the VD-deficient group (Figures 5 and 6A), TGF-β treatment further increased fibronectin and β-catenin concentrations, an effect that was blocked with VD pretreatment. Values are means ± SEM. N = 3. #P < 0.05 and *P < 0.05.

Figure 7.

Figure 7.

Perinatal VD status modulates LMSC paracrine effect on alveolar type II (ATII) cell proliferation and differentiation. (A) Primary Embryonic Day 19 fetal rat lung ATII cells were exposed to 1× , 1/5× , or 1/10× dilutions of conditioned media obtained from LMSCs from various VD-supplemented groups. Compared with the control group, ATII cells from all groups, exposed to 1× conditioned media, demonstrated significantly inhibited proliferation; however, compared with the VD-deficient group, there was amelioration of the inhibition in ATII cell proliferation in the 250 VD–, 500 VD–, and 1,000 VD–supplemented groups. Values are means ± SEM; N = 6 for each group. *P < 0.01 and **P < 0.001 (vs. control). #P < 0.01 (vs. VD-deficient group). (B) Compared with the control group, concentrations of SP-B (surfactant protein-B) and SP-C (surfactant protein-C) decreased significantly in the VD-deficient and 1,000 VD–supplemented groups, whereas these concentrations were close to those of the control group in the 250 VD– and 500 VD–supplemented groups. Values are means ± SEM; N = 3 for each group. *P < 0.05 (vs. control group).

Comment in

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