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. 2024 Nov 15;14(1):28100.
doi: 10.1038/s41598-024-79892-0.

HIF-1α knockdown attenuates phenotypic transformation and oxidative stress induced by high salt in human aortic vascular smooth muscle cells

Wenbin Deng  1 , Shiqiong Huang  1 , Lisha Yu  1 , Bo Gao  2 , Yun Pan  2 , Xue Wang  1 , Lihua Li  3   4
Affiliations

HIF-1α knockdown attenuates phenotypic transformation and oxidative stress induced by high salt in human aortic vascular smooth muscle cells

Wenbin Deng et al. Sci Rep. .

Abstract

Increased dietary salt intake is a well-established risk factor for hypertension and related cardiovascular diseases, involving complex vascular remodeling processes. However, the specific role of hypoxia-inducible factor-1α (HIF-1α) in vascular pathophysiology under high-salt conditions remains poorly understood. This study investigates the role of HIF-1α in high-salt-induced vascular remodeling using human aortic vascular smooth muscle cells (HA-VSMCs) cultured in vitro. HA-VSMCs were divided into three groups: high-salt with HIF-1α knockdown (shHIF-1α + HS), negative control (shcontrol), and high-salt (HS). Cell viability, migration, gene expression, and protein levels were evaluated. High-salt conditions significantly increased mRNA expression of α-smooth muscle actin (α-SMA), smooth muscle protein 22 (SM22), angiotensin II type 1 receptor (AT1R), collagen I, and collagen III (p < 0.0001). HIF-1α knockdown partially attenuated these increases, particularly for α-SMA, SM22, and AT1R (p < 0.01). At the protein level, high-salt exposure markedly elevated expression of collagen III, HIF-1α, osteopontin (OPN), and angiotensin II (Ang II) (p < 0.0001). HIF-1α knockdown significantly reduced the high-salt-induced increases in collagen III and HIF-1α protein levels (p < 0.001) but had a limited effect on OPN and Ang II upregulation. Interestingly, SM22 protein expression was significantly decreased under high-salt conditions (p < 0.0001), an effect partially reversed by HIF-1α knockdown (p < 0.0001). These findings demonstrate that high-salt conditions induce complex changes in gene and protein expression in HA-VSMCs, with HIF-1α playing a crucial role in mediating many of these alterations. The study highlights the differential effects of HIF-1α on various markers of vascular remodeling and suggests that HIF-1α may be a potential therapeutic _target for mitigating salt-induced vascular pathology. Further research is warranted to elucidate the mechanisms underlying the HIF-1α-dependent and -independent effects observed in this study.

Keywords: Angiotensin II; Human aortic vascular smooth muscle cell; Hypoxia-inducible factor-1α; Salt; Vascular remodeling.

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Conflict of interest statement

Declarations Competing interests The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Validation of HIF-1α knockdown efficacy in HA-VSMCs using RNA interference. (A, B) Representative fluorescence microscopy images (100× magnification) illustrating the transduction efficiency of lentiviral vectors in HA-VSMCs. Cells were transduced with either control shRNA (shcontrol) (A) or HIF-1α-_targeted shRNA (shHIF-1α) (B) constructs. Green fluorescence indicates successful transduction. Scale bars: 100 μm. (C) Quantitative assessment of HIF-1α mRNA expression levels in HA-VSMCs following lentiviral transduction. Expression was analyzed using real-time quantitative PCR (RT-qPCR) and normalized to the shcontrol group. Data are presented as mean ± standard deviation. Statistical significance was determined using Student’s t-test. ****P < 0.0001 compared to the shcontrol group.
Fig. 2
Fig. 2
Effects of elevated sodium concentrations on HA-VSMCs viability and the role of HIF-1α. (A) Time- and dose-dependent effects of varying sodium concentrations on HA-VSMC viability. Cells were exposed to a range of sodium concentrations (132–167 mmol/L) for 24, 48, 72, and 96 h. Cell viability was assessed and expressed as a percentage. (B) Comparative analysis of HA-VSMCs viability under high-sodium conditions in shcontrol, HS, and shHIF-1α + HS groups at 72 h post-treatment. Data are presented as mean ± standard deviation. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the shcontrol group. shcontrol, negative control group; HS, high salt group (152 mmol/L Na+); shHIF-1α + HS, high salt knockdown group treated with high salt; HA-VSMCs, human aortic vascular smooth muscle cell; HIF-1α, hypoxia-inducible factor 1-alpha.
Fig. 3
Fig. 3
High-salt stimulation promotes migration of HA-VSMCs, partially mediated by HIF-1α. (A) Representative images of the scratch wound healing assay for HA-VSMCs under different conditions. Images were taken at 0 h and 6 h after scratch formation. Yellow lines indicate the edges of the scratch wound. (B) Quantification of cell motility expressed as cell mobility (%). Compared to the shcontrol group, both the high salt (HS) and HIF-1α knockdown (shHIF-1α + HS) groups showed significantly increased cell mobility. The HS group demonstrated the highest mobility, while the shHIF-1α + HS group showed an intermediate level of mobility between shcontrol and HS. ****P < 0.0001; *** P < 0.001. Scale bar: 200 μm.
Fig. 4
Fig. 4
High-salt stimulation induces changes in oxidative stress markers and OPN expression in HA-VSMCs. (A–C) Quantitative analysis of (A) SOD activity (U/mgprot), (B) MDA levels (nmol/mgprot), and (C) OPN expression (ng/ml) in negative control (shcontrol), high salt (HS), and high-salt with HIF-1α knockdown (shHIF-1α + HS) groups. Data are presented as mean ± standard deviation. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001. SOD, superoxide dismutase; MDA, malondialdehyde; OPN, osteopontin.
Fig. 5
Fig. 5
mRNA expression levels of key genes in response to high salt and HIF-1α knockdown. (A–F) Quantitative analysis of mRNA expression levels for various genes in negative control (shcontrol), high salt (HS), and high-salt with HIF-1α knockdown (shHIF-1α + HS) groups. (A) α-SMA, (B) SM22, (C) Collagen I, (D) Collagen III, (E) HIF-1α, and (F) AT1R. Data are presented as mean ± standard deviation. ns: not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001. α-SMA, alpha-Smooth Muscle Actin; SM22, Smooth Muscle Protein 22-alpha; HIF-1α: Hypoxia-Inducible Factor 1-alpha; AT1R: Angiotensin II Type 1 Receptor.
Fig. 6
Fig. 6
Expression levels of key proteins in different experimental groups. (A) Representative western blot images showing the protein expression of Collagen III, HIF-1α, OPN, Ang II, SM22, and β-actin in the negative control (shcontrol), high salt (HS), and high-salt with HIF-1α knockdown (shHIF-1α + HS) groups. (B–F) Quantitative analysis of protein expression levels normalized to β-actin. (B) Collagen III, (C) HIF-1α, (D) OPN, (E) Ang II, and (F) SM22. Data are presented as mean ± standard deviation. n = 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. HIF-1α: Hypoxia-Inducible Factor 1-alpha; OPN, Osteopontin; Ang II, Angiotensin II; SM22, Smooth Muscle Protein 22-alpha.
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