In patients with pulmonary arterial hypertension (PAH), zinc homeostasis plays a key protective role in the pathogenesis of the disease.
Thus, regulation of metal responsive transcription factor 1 (MTF-1)—a zinc-dependent transcription factor—may be a potential target for therapeutic intervention in PAH, according to findings from a study published in the journal Cell Cycle.
Individuals with PAH experience a progressive increase in mean pulmonary arterial pressure, remodeling of their pulmonary vasculature, and right heart failure. The aberrant proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) are the main pathologic bases for this remodeling.
The researchers of the current analysis sought to explore the underlying mechanisms and effects of MTF-1 on the proliferation of PASMCs in patients with PAH. The involvement of zinc in PAH was established by evaluating the proliferation of PASMCs after injecting monocrotaline (MCT) and administering zinc sulfate (ZnSO4) in rats. An additional examination was performed to determine whether zinc activates MTF-1 in PASMCs via upregulation of its expression and transcriptional activity.
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Twenty-two male Sprague-Dawley rats, which weighed between 150 and 180 grams, were randomly allocated to 1 of
2 groups: the MCT-PAH group and the control group. The MCT-PAH rats received an intraperitoneal injection of 20 mg/kg of MCT on days 1 and 8, whereas the control rats received the same volume of saline.
The following techniques were used to perform a comprehensive analysis of MTF-1 in the proliferation of PASMCs in PAH:
- Histologic and immunohistochemical analysis
- Fluorescent immunohistochemistry
- Cell culture and treatment
- Generation of polyclonal anti-MTF-1 antibody
- Cell immunofluorescence staining
- Cell proliferation assay
- Cell scratch assay
- Metal transcription factor 1 (Mtf-1)/placental growth factor (Plgf)-short interfering (si)RNA transfection
- Dual-luciferase reporter assay
- Measurement of free intracellular zinc with the fluorescent indicator fluozin-3-AM
- Cytoplasmic and nuclear protein extraction
- Protein extraction and Western blot
Results of the study showed that the relative fluorescence intensity of fluozin-3-AM was higher in the PASMCs isolated from the MCT-PAH arm than in those from the control arm. In addition, increased intracellular-free zinc ions were revealed in the MCT-PASMCs and ZnSO4-supplemented PASMCs, leading to overexpression and overactivation of MTF-1, along with upregulation of placental growth factor.
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Elevated levels of MTF-1 and placental growth factor were seen on Western blot; high transcriptional activity of MTF-1 was corroborated by luciferase reporter assay in ZnSO4-treated PASMCs. Based on flow cytometry evaluation, blockade of protein kinase C signaling inhibited the cell cycle of MCT-PASMCs, as well as ZnSO4-enhanced PASMCs.
Further, MCT-induced PAH was associated with significant elevations in mean pulmonary arterial pressure and right ventricular hypertrophy index (P <.05). Assessment of cell proliferation demonstrated a favorable effect of zinc ions on proliferation of PASMCs, with deletion of Mtf-1/Plgf decreasing ZnSO4-generated proliferation.
“Our findings support the notion that local zinc ion depletion, suppression of zinc ion influx, and inhibition of MTF-1 expression and activity in lung tissue would be effective therapeutic approaches for the treatment of PAH,” the researchers concluded.
Chen A, Gao G, Lian G, et al. Zinc promotes cell proliferation via regulating metal-regulatory transcription factor 1 expression and transcriptional activity in pulmonary arterial hypertension. Cell Cycle. Published online May 1, 2023. doi:10.1080/15384101.2023.2205209