Megakaryocytic leukemia 1 (MKL1) regulates hypoxia induced pulmonary hypertension in rats

Abstract

Hypoxia induced pulmonary hypertension (HPH) represents a complex pathology that involves active vascular remodeling, loss of vascular tone, enhanced pulmonary inflammation, and increased deposition of extracellular matrix proteins. Megakaryocytic leukemia 1 (MKL1) is a transcriptional regulator known to influence cellular response to stress signals in the vasculature. We report here that in response to chronic hypobaric hypoxia, MKL1 expression was up-regulated in the lungs in rats. Short hairpin RNA (shRNA) mediated depletion of MKL1 significantly ameliorated the elevation of pulmonary arterial pressure in vivo with a marked alleviation of vascular remodeling. MKL1 silencing also restored the expression of NO, a key vasoactive molecule necessary for the maintenance of vascular tone. In addition, hypoxia induced pulmonary inflammation was dampened in the absence of MKL1 as evidenced by normalized levels of pro-inflammatory cytokines and chemokines as well as reduced infiltration of pro-inflammatory immune cells in the lungs. Of note, MKL1 knockdown attenuated fibrogenesis in the lungs as indicated by picrosirius red staining. Finally, we demonstrate that MKL1 mediated transcriptional activation of type I collagen genes in smooth muscle cells under hypoxic conditions. In conclusion, we data highlight a previously unidentified role for MKL1 in the pathogenesis of HPH and as such lay down groundwork for future investigation and drug development.

Figure 1. MKL1 expression is up-regulated in the lungs in rats with hypoxia-induced pulmonary hypertension.

Sprague Dawley rats were injected with lentiviral particles carrying shRNA targeting MKL1 or random shRNA (SCR) and induced to develop HPH as described under Methods. (A) MKL1 mRNA and protein levels in pulmonary arteries were assessed by qPCR and Western blotting. (B) MKL1 and α-SMA levels in pulmonary arteries were examined by immunohistochemistry. (C) Protein expression of MKL1 and α-SMA was quantified by Image Pro and expressed as relative staining compared to the control group set as 1. N = 10 rats for each group.

Figure 2. MKL1 silencing attenuates hypoxia-induced pulmonary hypertension in rats.

Sprague Dawley rats were injected with lentiviral particles carrying shRNA targeting MKL1 or random shRNA (SCR) and induced to develop HPH as described under Methods. (A) Pulmonary arterial pressure was measured. N = 10 rats for each group (B) RV/(LV+SV) was measured to assess right ventricle hypertrophy. N = 10 rats for each group (C, D) Immunofluorescence staining was performed with anti-α-SMA (orange) and anti-VWF (green). Medial thickness and lumen diameter were measured by Image J. N = 10 rats for each group (E, F) Histochemical stainings were performed as described under Methods. Muscularization of pulmonary vessels was evaluated as described in Methods. N = 10 rats for each group (G) Levels of NO were evaluated by a commercially available kit as described under Methods. N = 5 rats for each group.

Figure 3. MKL1 silencing attenuates hypoxia-induced pulmonary inflammation in rats.

Sprague Dawley rats were injected with lentiviral particles carrying shRNA targeting MKL1 or random shRNA (SCR) and induced to develop HPH as described under Methods. (A, B) Levels of cytokines and chemokines were assessed by ELISA. N = 5 rats for each group (C, D) Immunohistochemistry was performed with indicated antibodies as described under Methods and quantified by Image J. N = 5 rats for each group.

Figure 4. MKL1 silencing attenuates hypoxia-induced pulmonary fibrogenesis in rats.

Sprague Dawley rats were injected with lentiviral particles carrying shRNA targeting MKL1 or random shRNA (SCR) and induced to develop HPH as described under Methods. (A) Picrosirius red staining was performed as described under Methods. (B) Positive staining was quantified by Image J and expressed as relative fibrosis compared to the control group. A. U., arbitrary unit (C, D) Expression of molecules involved in fibrogenesis in pulmonary arteries was measured by qPCR (C) and Western blotting (D). N = 5 rats for each group (E) Levels of TGF-β in pulmonary arteries were assessed by ELISA. N = 4 rats for each group.

Figure 5. MKL1 regulates hypoxia-induced collagen transcription in smooth muscle cells.

(A) Col1a1 or col1a2 promoter luciferase construct was transfected into A10 cells with MKL1 expression construct followed by exposure to 1% O2 for 24 hours. Luciferase activities were expressed as relative luciferase unit (RLU). (B) Col1a1 or col1a2 promoter luciferase construct was transfected into A10 cells with shRNA plasmid targeting MKL1 or a control shRNA plasmid (nontarget) followed by exposure to 1% O2 for 24 hours. Luciferase activities were expressed as RLU. (C, D) A10 (C) or primary human pulmonary arterial smooth muscle (D) cells were transfected with MKL1 specific or control siRNA (SCR) followed by exposure to 1% O2 for 24 hours. Expression of collagen type I genes was measured by qPCR.