Hypoxia-Induced Mitogenic Factor (HIMF/FIZZ1/RELMα) Recruits Bone Marrow-Derived Cells to the Murine Pulmonary Vasculature

. and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs). The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner.These results demonstrate HIMF-dependent recruitment of BMD mesenchymal-like cells to the remodeling pulmonary vasculature

HIMF expression in murine lung.

<p>Paraffin-embedded lung sections from normoxic (7d, 20.8% O₂) (A), hypoxic (7d, 10.0% O₂) (B), AAV-null-treated (14d, 2.5×10¹⁰ VP) (C), and AAV-HIMF-treated (14d, 2.5×10¹⁰ VP) (D) mice were rehydrated and stained with goat anti-mouse HIMF polyclonal antibodies. Pa: pulmonary artery. Aw: airway. Scale bar: 50 µm.</p

Both chronic hypoxia and pulmonary HIMF gene transfer recruit BMD cells to the pulmonary vasculature.

<p>(A–C, J–M) Light micrograph of fluorescence images to show blood vessel structure. Frozen sections from normoxic (20.8% O₂) (D, N), hypoxic (10.0% O₂) (E, O), and AAV-HIMF treated (2.5×10¹⁰ VP) (F, P, Q) lungs were stained with a rabbit anti-GFP polyclonal antibody that was visualized by an FITC-conjugated goat anti-rabbit IgG antibody (green). (G–I, R–U): Differential interference contrast images of light and fluorescence images to show structure. A–L, N–P, R–T: Scale bar: 50µm. M, Q, U: Scale bar: 20µm.</p

GFP and cellular markers sca-1 and c-kit co-localize in AAV-HIMF-treated bone marrow transplant recipients.

<p>Frozen lung sections from bone marrow transplant recipients treated with AAV-HIMF (2.5×10¹⁰ VP, 14d) were stained with antibodies for (B) sca-1, (F) c-kit, or (J) CD34 (red). (C, G, K) GFP signal was obtained through direct visualization (green). (A, E, I) Cell nuclei were counterstained with DAPI (blue). (D, H, L) The arrows in the merged images demonstrate co-localization of GFP with the cellular markers. Scale bar: 20 µm.</p

Chronic hypoxia and pulmonary HIMF gene transfer increase the number of BMD cells associated with the pulmonary vasculature.

<p>A–D: Paraffin-embedded lung sections from mice exposed to normoxia (7d, 20.8% O₂) (A), hypoxia (7d, 10.0% O₂) (B), AAV-null (14d, 2.5×10¹⁰ VP) (C), or AAV-HIMF (14d, 2.5×10¹⁰ VP) (D) were probed with polyclonal antibodies raised against GFP. Arrows indicate GFP⁺ cells within the vasculature. Scale bar: 50 µm. E, F: Quantification of GFP⁺ cells within the pulmonary vasculature. GFP⁺ cells within the pulmonary vasculature are shown as mean ± SEM of GFP⁺ cells/vessel. * <i>P</i><0.05, ** <i>P</i><0.01 vs. control.</p

Both chronic hypoxia and pulmonary HIMF gene transfer induce pulmonary vascular remodeling.

<p>A–D: Paraffin-embedded lung sections were double-stained with antibodies to von Willebrand factor (black) and α-smooth muscle actin (red). Arrows indicate small pulmonary vessels. Scale bar: 50 µm. E, F: Percent muscularization of small pulmonary arteries in mouse lungs. NM, non-muscularized; PM, partially muscularized; FM, fully muscularized. *Significantly decreased vs. control at <i>P</i><0.05. ^†Significantly increased vs. control at <i>P</i><0.05.</p

GFP⁺ cells were recruited to the smooth muscle layer of the pulmonary vasculature in AAV-HIMF-treated mice.

<p>GFP was detected through direct visualization (A, E; green). HIMF and α-smooth muscle actin (α-SMA) were detected with anti-HIMF and anti-α-SMA primary antibodies and visualized with rhodamine-conjugated anti-rabbit IgG secondary antibodies (B; red) and Cy5-conjugated anti-mouse IgG secondary antibodies (C; pink), respectively. Arrows in the merged image indicate co-localization of GFP, HIMF, and α-SMA (D). Lung sections were stained with anti-CD31 antibodies and visualized with rhodamine-conjugated anti-rat IgG antibodies (F; red) and anti-α-SMA antibodies and visualized with Cy5-conjugated anti-mouse IgG antibodies (G, pink). (F) Arrows in the merged image indicate co-localization of GFP and α-SMA. Cell nuclei were stained with DAPI (D, H; blue) Scale bar: 20 µm.</p