Modulation of bleomycin-induced lung fibrosis by pegylated hyaluronidase and dopamine receptor antagonist in mice

Abstract

Hyaluronidases are groups of enzymes that degrade hyaluronic acid (HA). To stop enzymatic hydrolysis we modified testicular hyaluronidase (HYAL) by activated polyethylene oxide with the help of electron-beam synthesis. As a result we received pegylated hyaluronidase (pegHYAL). Spiperone is a selective D2 dopamine receptor antagonist. It was demonstrated on the model of a single bleomycin damage of alveolar epithelium that during the inflammatory phase monotherapy by pegHYAL or spiperone reduced the populations of hematopoietic stem /progenitor cells in the lung parenchyma. PegHYAL also reduced the levels of transforming growth factor (TGF)-β, interleukin (IL)-1β, tumor necrosis factor (TNF)-α in the serum and lungs, while spiperone reduced the level of the serum IL-1β. Polytherapy by spiperone and pegHYAL caused the increase of the quantity of hematopoietic stem/ progenitor cells in the lungs. Such an influx of blood cell precursors was observed on the background of considerable fall level of TGF-β and the increase level of TNF-α in the serum and lungs. These results show pegHYAL reduced the bleomycin-induced fibrosis reaction (production and accumulation of collagen) in the lung parenchyma. This effect was observed at a single and repetitive bleomycin damage of alveolar epithelium, the antifibrotic activity of pegHYAL surpassing the activity of testicular HYAL. The antifibrotic effect of pegHYAL is enhanced by an additional instillation of spiperone. Therapy by pegHYAL causes the flow of CD31‒ CD34‒ CD45‒ CD44+ CD73+ CD90+ CD106+-cells into the fibrous lungs. These cells are incapable of differentiating into fibroblast cells. Spiperone instillation separately or together with pegHYAL reduced the MSC-like cells considerably. These data enable us to assume, that pegHYAL is a new and promising instrument both for preventive and therapy of toxic pneumofibrosis. The blockage of D2 dopamine receptors with the following change of hyaluronan matrix can be considered as a new strategy in treatment of pneumofibrosis.

Fig 1. Effect of bleomycin on the lung architecture in C57Bl/6 mice.

Comparison of the lung architecture in C57Bl/6 mice after BLM instillation (21st day of the experiment—partially reversible pulmonary fibrosis or 60th day of the experiment—irreversible pulmonary fibrosis) or 0.9% NaCl (sham treatment), as shown by hematoxylin-eosin (A) and picrofuchsin (B) staining of representative tissue sections. The photomicrographs were taken using an Axio Lab.A1 (Carl Zeiss MicroImaging GmbH; Göttingen, Germany) microscope and AxioCam ERc5s digital camera. All photomicrographs were at 100 × magnification.

Fig 2. Photomicrographs of representative lung sections obtained from C57BL/6 mice after a single bleomycin instillation (stained by hematoxylin-eosin).

Tissues were stained with hematoxylin-eosin to investigate inflammatory cells accumulation (21st day of the experiment). (A) Mice receiving intratracheal 0.9% NaCl, (B) Mice receiving intratracheal BLM, (C) Mice with fibrosis spiperone treated, (D) Mice with fibrosis HYAL treated, (E) Mice with fibrosis pegHYAL treated, (F) Mice with fibrosis pegHYAL and spiperone treated. The photomicrographs were taken using an Axio Lab.A1 (Carl Zeiss MicroImaging GmbH; Göttingen, Germany) microscope and AxioCam ERc5s digital camera. All photomicrographs were at 100 × magnification.

Fig 3. Photomicrographs of representative lung sections obtained from C57BL/6 mice after a single bleomycin instillation (stained by picrofuchsin).

Tissues were stained with picrofuchsin to determine the collagen content (21st day of the experiment). (A) Mice receiving intratracheal 0.9% NaCl; (B) Mice receiving intratracheal BLM; (C) Mice with fibrosis spiperone treated; (D) Mice with fibrosis HYAL treated; (E) Mice with fibrosis pegHYAL treated; (F) Mice with fibrosis pegHYAL and spiperone treated. Dark pink stains are collagenous deposits. The most expressed collagen fibers deposition after BLM injection was observed on the 21st day. The photomicrographs were taken using an Axio Lab.A1 (Carl Zeiss MicroImaging GmbH; Göttingen, Germany) microscope and AxioCam ERc5s digital camera. All photomicrographs were at 100 × magnification.

Fig 4. Characterization of hematopoietic stem cells isolated from lung of C57BL/6 mice (7th day of the experiment).

The phenotype of cells from lung was studied according to the protocol for hematopoietic stem cells (BD Biosciences). The HSC population taken through a Lin^- selection (not shown) and then Sca1⁺ and c-kit⁺ (not shown), is shown gated displayed for CD34^- (P7) and CD34⁺ (P8). The Lin^-Sca1⁺c-kit⁺CD34^- cells are LT-HSCs and the Lin^-Sca1⁺c-kit⁺CD34⁺ cells are considered ST-HSCs. Thus, all two of these populations can be readily sorted from one sample. (A) HSCs isolated from mice after intratracheal 0.9% NaCl administration; (B) HSCs isolated from mice after intratracheal BLM administration; (C) HSCs isolated from mice after intratracheal BLM administration and treated spiperone; (D) HSCs isolated from mice after intratracheal BLM administration and treated pegHYAL; (E) HSCs isolated from mice after intratracheal BLM administration and treated spiperone and pegHYAL; (F) HSCs isolated from mice after intratracheal BLM administration and treated HYAL. Dot plots are representative figures of three independent experiments with the mean from three independent experiments.

Fig 5. Analysis of murine pan-hematopoietic stem cells (CD45⁺-cells) and CD45^- -cells derived from lung (21^st day of the experiment).

Cells were analyzed by FACS with antibody to identify mouse CD45, CD90.1, CD34, CD73, CD106 (VCAM-1), CD44 and CD31 (PECAM-1) (BD Biosciences, USA). Cells for analysis were derived from lungs of C57BL/6 mice of control and experimental groups. The CD45 negative cells population and CD45 positive cells population were sorted from one sample. The CD45 negative cells population was defined as CD34 and CD31 negative and positive for CD106, CD73, CD44, and CD90.1 at the same time in the two specimens (1 specimen—subpopulation of cells with phenotype CD45^-, CD73⁺, CD90⁺, CD106⁺, CD44⁺ and 2 specimen—subpopulation of cells with phenotype CD45^-, CD73⁺, CD90⁺, CD31^-, CD34^-). Dot plots are representative figures of three independent experiments with the mean from three independent experiments.