Calpain‐1 mediates vascular remodelling and fibrosis via HIF‐1α in hypoxia‐induced pulmonary hypertension

Abstract Calpain‐1, a calcium‐activated neutral cysteine proteases, has been reported to be involved in the formation of pulmonary hypertension. HIF‐1α, an oxygen‐sensitive transcription factor, has been reported to activate genes involved in cell proliferation and extracellular matrix recombination. This study was designed to investigate the effect of calpain‐1 in hypoxic pulmonary hypertension (HPH) and to explore whether there is a relationship between calpain‐1 and HIF‐1α in this disease. In the hypoxia‐induced model of HPH, we found that hypoxia resulted in increased right ventricular systolic pressure, right ventricular hypertrophy, pulmonary vascular remodelling and collagen deposition in lung tissues of mice. The levels of calpain‐1 and HIF‐1α were up‐regulated in the lung tissues of hypoxia‐treated mice and pulmonary arterial smooth muscle cells (PASMCs). Knock‐out of calpain‐1 restrained haemodynamic and histological changes induced by chronic hypoxia in mice, and inhibition of calpain‐1 also repressed the abnormal proliferation and migration of PASMCs. Besides, knock‐out or inhibition of calpain‐1 suppressed hypoxia‐induced expression of HIF‐1α, VEGF, PCNA, TGF‐β1, MMP2 and collagen I in vivo and in vitro. While inhibition of HIF‐1α abolished the above effects of calpain‐1. Furthermore, we found that calpain‐1 mediates the expression of HIF‐1α through NF‐κB (P65) under hypoxia conditions. In conclusion, our results suggest that calpain‐1 plays a pivotal role in hypoxia‐induced pulmonary vascular remodelling and fibrosis through HIF‐1α, providing a better understanding of the pathogenesis of HPH.

artery pressure and finally lead to right heart failure and death. 3,4 Up to now, the exact pathogenesis of HPH is still unclear, and there is a lack of effective treatment in clinic. Therefore, it is necessary to find new potential therapeutic targets for HPH.
The calpain system has been discovered for more than 50 years and is expressed in multiple organs of human body. 5 Calpains belong to the calcium-dependent non-lysosomal neutrocysteine endopeptidyase family and include 15 isoforms. Among them, calpain-1 and calpain-2 are two major typical calpain proteinases, which are composed of 80 kD large subunits with catalytic activity and 28 kD small subunits with regulatory activity. 6,7 Calpains have been found to be involved in the pathological process of pulmonary hypertension (PH). For example, Wan et al 8 . showed that the imbalance of extracellular calpain/calpastatin is involved in the pathological process of PH. In addition, Zhang et al 9 . found that calpain-1 expression is significantly up-regulated in rat pulmonary arteries under hypoxic conditions, which promotes abnormal proliferation of PASMCs and participates in the development of HPH. However, the mechanism of how calpain induces the proliferation of PASMCs has not been clarified. In addition, calpain has been found to mediate the synthesis of Collagen I by promoting the synthesis and activation of TGF-β1 in bleomycin-induced pulmonary fibrosis. 10 Hypoxia-inducible factor-1 (HIF-1) is composed of the following two subunits: a stably expressed β subunit and an O 2 -regulated α subunit. Under low oxygen conditions, HIF-1α accumulates and transferred to the nucleus, combines with HIF-1β to form a heterodimer, transcribes and activates genes related to energy metabolism, cell proliferation, apoptosis and extracellular matrix recombination. 11, 12 Mo et al 13 . found that HIF-1α overexpression increases VEGF expression and calpain activity in human umbilical vein endothelial cells, leading to angiogenesis. Besides, studies have shown that HIF-1α is critical for TGF-β1-induced Collagen I synthesis in glomerulosclerosis. 14 Previous studies in our lab have shown that calpain-1 mediates IκBα degradation and NF-κB activation in heart tissue. 15 In addition, studies have shown that P65 is one of the key transcription factors in PASMCs that promote HIF-1α protein accumulation. 16 According to the above research status, we hypothesized that calpain-1 is involved in hypoxia-induced pulmonary vascular remodelling and fibrosis in a HIF-1α dependent manner, hoping to provide a new theoretical basis for calpain-1 as a potential therapeutic target of HPH.

| Animal experiments
Animal experiments were conducted in accordance with the guidelines of Animal Care and Use Committee of Jinzhou Medical University (2021018). C57BL/6 mice were acquired from Liaoning changsheng biotechnology co. LTD. Calpain-1(−/−) C57BL/6 mice were obtained from Cyagen (Guangzhou) Biotechnology Co. LTD.
The mice were randomly assigned to the following six groups (n = 8 per group): WT Normoxia, KO Normoxia, WT Hypoxia, KO Hypoxia, Hypoxia+YC-1 and Hypoxia+BAY11-7082. The mice in hypoxia+YC-1 and hypoxia+BAY11-7082 groups were separately intraperitoneal injection with YC-1 (1 mg/kg/day, Selleck) and BAY11-7082 (5 mg/kg, 3 times/week, Selleck). The mice in normoxia groups were exposed to normal environment containing 21% O 2 , while those in hypoxia groups were exposed to a normobaric chamber containing 10% O 2 for 4 weeks. 17 After 4 weeks, the mice were anaesthetized with 1% pentobarbital sodium (40 mg/kg i.p.). Carefully detach the right external jugular vein and the return blood flow was blocked with a fine line. Then, the Miller catheter connected to the biofunctional experimental system was slowly inserted through an incision cut in the right external jugular vein to measure right ventricular systolic pressure (RVSP). 18 The collected serum and cardiopulmonary tissues were stored in a −80°C refrigerator for further experiments.

| Culture and treatment of primary rat PASMCs
SD rats were anesthetized with pentobarbital sodium and soaked with 75% alcohol for disinfection. The pulmonary arteries were then isolated in a sterile environment. After scraping the inner and outer membranes, the middle membranes were cut into small pieces and transferred to cell culture vials. DMEM (Gibco) supplemented with 20% foetal bovine serum (FBS, Sigma) and 1% penicillin/streptomycin was added and cultured in an incubator (37°C, 5% CO 2 ). After 3-5 days, cells can be observed around the tissue mass.

| Histology analysis
The lungs of mice in each group were fixed with 4% paraformaldehyde for 48 h, embedded in paraffin after gradient ethanol, and cut into 4μm-thick sections for further experiment. The paraffin sections were stained by haematoxylin-eosin (HE) staining and Masson staining, respectively, to observe vascular remodelling and fibrosis.
The percentage of pulmonary artery wall thickness to total thickness (WT/TT)% and the percentage of wall area to total area (WA/TA)% were calculated and analysed by Image-Pro Plus to evaluate pulmonary artery remodelling. 3

| Determination of right ventricular hypertrophy
The hearts excised from the mice were washed with PBS and divided into two parts: right ventricle (RV) and left ventricle plus interventricular septum (LV+S), and their dry weights were weighed, respectively. The degree of right cardiac hypertrophy was calculated and evaluated according to right ventricular hypertrophy index: (RV/ [LV+S]). 19

| Immunohistochemical staining
After routine dewaxing and hydration, the sections were subjected to high-pressure antigen repair, and then incubated with 3% H 2 O 2 , blocked with goat serum and incubated overnight with primary antibody Ki-67 (1:100, Beyotime) at 4°C. The next day, the second antibody was incubated in darkness, and then DAB (ZSGB-BIO) was used to visualize, observed and interpreted under light microscope.

| Immunofluorescence staining
Pulmonary arterial smooth muscle cells cultured in 24-well plates were fixed with 4% paraformaldehyde dissolved in PBS and then permeabilized with 0.3% triton X-100. After blocking with 5% BSA, pri-

| Western blot assay
Lung tissues and PASMCs were lysed in radio immunoprecipitation assay (RIPA) lysis buffer (containing 1% PMSF) on ice to extract total proteins. Nuclear and cytoplasmic proteins were extracted following the manufacturer's recommendations of the nuclear and cytoplasmic extraction kit (Beyotime). After protein quantification, the same amount of protein from each sample were separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then the proteins on the gel were transferred to PVDF membranes (Millipore) by semi-dry electrophoretic transfer system (Bio-rad).

| ELISA assay
TGF-β1 levels in the serum of mice were determined with a mouse TGF-β1 ELISA kit (mlbio) following the manufacturer's recommendations.

| Cell proliferation and migration assay
Pulmonary arterial smooth muscle cells were inoculated into 96well plates and cultured in hypoxic or normoxic incubators for 24 h.

| Statistical analysis
All data were presented as means ± SD, and GraphPad Prism (version 8.0.2) was used for statistical analysis. One-way analysis of variance (ANOVA) was used to test the statistical differences among the experimental groups. p < 0.05 was statistically significant.

| Expression of calpain-1 in mice model of HPH
To demonstrate whether calpain-1 is involved in the pathogenesis of HPH, Western blot analysis of lung tissues was performed to assess calpain-1 protein levels in C57BL/6 mice treated with hypoxia or normoxia for 4 weeks. As illustrated in Figure 1A,B, compared with normoxic group, calpain-1 levels in the lung tissues of hypoxic mice were significantly increased. To reduce the level of calpain-1, calpain-1 was knocked out in mice. Compared with WT mice, the calpain-1 level of KO mice was significantly decreased in lung tissues.

| Calpain-1 is involved in hypoxia-induced pulmonary vascular remodelling and fibrosis
Haemodynamic and vascular remodelling parameters were measured in C57BL/6 mice treated with hypoxia or normoxia for 4 weeks. As shown in Figure 2A

| Effects of calpain-1 on proliferation and migration of hypoxia-treated PASMCs
Primary PASMCs were extracted from rat pulmonary arteries and identified by α-SMA ( Figure 3A), then exposure to hypoxia. Figure 3B-D, hypoxia treatment significantly increased calpain-1 levels in PASMCs, while calpain-1 levels were significantly inhibited by calpain-1 inhibitor MDL28170. As shown in Figure 3E-G, the proliferation and migration of PASMCs were detected by CCK-8 and transwell assays respectively. The results showed that the proliferation and migration of PASMCs were significantly enhanced after hypoxia, while MDL28170 inhibited them.

| Calpain-1 promoted the expression of proliferation and fibrosis-related molecules through HIF-1α
We

| Calpain-1 mediates the expression of HIF-1α through NF-κB under hypoxia conditions
Based on the observation that calpain-1 regulates the protein expression of HIF-1α, we further explored the mechanism of how calpain-1 triggers HIF-1α expression in lung tissues and PASMCs.
Previous studies in our laboratory have shown that calpain-1 promotes the activation of NF-κB in heart tissue. In addition, studies have shown that NF-κB is one of the key transcription factors in PASMCs that promote HIF-1α protein accumulation. We next investigated whether NF-κB (P65) is involved in calpain-1 mediated HIF-1α expression in HPH mice lung tissues and PASMCs under hypoxia. As shown in Figure 6A-C, hypoxia can lead to the increased expression of nuclear protein P65 in the lung tissues of WT mice, but no significant changes were observed in KO mice. Besides, inhibition of NF-κB expression by BAY11-7082 attenuated the promotion effect of calpain-1 on HIF-1α expression ( Figure 6D,E). Meanwhile, the same results were observed in PASMCs ( Figure 6F-K).

| A positive feedback loop exists between calpain-1-HIF-1α axes during hypoxia
To further explore the relationship between calpain-1 and HIF-1α, the expression of HIF-1α in PASMCs was inhibited by YC-1. IF and Western blot results indicated that inhibition of HIF-1α decreased hypoxia-induced calpain-1 expression ( Figure 3B and Figure 7A,B).
Besides, pharmacological inhibition of HIF-1α signalling pathway also attenuated the effect of calpain-1 on nuclear protein P65 expression during hypoxia ( Figure 7C-E), indicating that HIF-1α is responsible for hypoxia and calpain-1-induced nuclear protein P65 secretion.
These results indicated that a positive feedback loop exists between HIF-1α and calpain-1/NF-κB signalling during hypoxia.

| DISCUSS ION
In this study, the mice were exposed to a normobaric chamber containing 10% O 2 for 4 weeks to develop a model of pulmonary hypertension as previously reported. 17 We found for the first time that cross-regulation between calpain-1 and HIF-1α is a key process that promotes the development of HPH. Furthermore, we found that calpain-1 interacts with HIF-1α in vivo and in vitro by activat- Studies have shown that activation of P65 leads to activation of HIF-1α promoter activity, which increases HIF-1α mRNA level and promotes HIF-1α protein accumulation. 16,35 Besides, overexpression of NF-κB also resulted in increased HIF-1α protein expression even under normoxic environments in human embryonic kidney (HEK)-293 cell. 36 In this study, we found that hypoxia resulted in an calpain-1-dependent increase in nuclear protein P65 expression. In addition, inhibition of NF-κB expression with BAY11-7082 significantly attenuated the promotion effect of calpain-1 on HIF-1α expression.
Further research found that inhibition of HIF-1α reduced the expression of calpain-1 induced by hypoxia, which also attenuated the effect of calpain-1 on nuclear protein P65 expression during hypoxia, indicating that a positive feedback loop exists between HIF-1α and calpain-1/NF-κB signalling during hypoxia.
In conclusion, our results provide experimental evidence that the calpain-1-HIF-1α axis plays a pivotal role in hypoxia-induced vascular remodelling and fibrosis, and provide a new theoretical basis for calpain-1 as a potential therapeutic target for HPH.

ACK N OWLED G EM ENTS
The author would like to thank professor Hongxin Wang, Meili Lu and the senior brothers and sisters of the laboratory. This work

CO N FLI C T O F I NTE R E S T
All authors claimed that there was no conflict of interest in the study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.