Upregulation of Nox4 in the aging vasculature and its association with smooth muscle cell polyploidy

Abstract

Our recent reports indicated that polyploidization of aortic vascular smooth muscle cells (VSMC) serves as a biomarker for aging, and that the polyploid state is linked to a higher incidence of senescence in vivo. Here, we found that NADPH oxidase 4 (Nox4) expression is augmented in VSMC from aortas of old rats and that Nox4 levels are increased in polyploid VSMC in comparison to diploid cells in vivo. Seeking to determine if Nox4 upregulation plays a causal role in the accumulation of polyploid cells, we performed ploidy analysis on primary VSMC transduced with Nox4 adenovirus. We observed a consistent accumulation of polyploid cells and a concomitant decrease in the percentage of diploid cells in Nox4 overexpressing cells in comparison to controls or to cells overexpressing dominant negative Nox4. Further exploration of this phenomenon in VSMC cultures identified a Nox4-induced decrease in the chromosome passenger protein, survivin, whose absence and mislocalization during polyploidization was previously shown to induce VSMC polyploidy. Taken together, our study is the first to show increased Nox4 levels in VSMC during aging, and to demonstrate its role in induction of polyploidy in this lineage.

Figure 1. Nox4 mRNA is upregulated in VSMC during aging and in polyploid VSMC in vivo

A. Nox4 RT-PCR of RNA from young and old aortas of Brown Norway (BN) rats. Age is indicated in months (m). GAPDH was used as a loading control. Data are representative of three experiments. A 3-4 fold increase in Nox4 mRNA was observed in 24 month old and 34 month old BN rats, as also depicted in Figure 2. Quantitation was performed using ImageJ 1.34s software (NIH, Bethesda, Maryland). B. Nox4 RT-PCR on 2N and 4N fractions of freshly isolated VSMC. VSMC were isolated from 12-month-old BN rats and then sorted by DNA content using flow cytometry (2N = diploid DNA content) as described under Methods. Nox4 transcript was upregulated approximately 2-fold in 4N fractions. Data are representative of two experiments each analyzed twice.

Figure 2. Nox4 protein is upregulated in VSMC during aging in vivo

Nox4 immunostaining (red) on aortic sections from 3 month, 24 month, and 32 month BN rats. Sections were counterstained with DAPI (blue) to visualize DNA in addition. Staining with IgG was used as a control.

Figure 3. Nox4 overexpression induces polyploidization of VSMC

A. Confirmation of overexpression of Nox4 and Nox4-ΔFAD cDNAs in transduced VSMC cultures. RT-PCR of Nox4/Nox4-ΔFAD transcripts from rat primary VSMC transduced with Ad-β-gal, Ad-Nox4, or Ad-Nox4-ΔFAD adenoviruses. GAPDH was used as a loading control in addition to reactions with no reverse transcriptase (RT) as a template control. B. Western blot of VSMC demonstrating overexpression of Nox4 by Ad-Nox4 transduction. Two Nox4 reactive bands were identified predominately in the transduced VSMC (this protein is known to be glycosylated) although basal Nox4 levels are also detectable. The antibody did not identify overexpression of truncated Nox4 in Ad-Nox4-ΔFAD treated VSMC because it recognizes an epitope in the deleted domains (not shown), however its expression was confirmed at the mRNA level as in A. C. β-galactosidase activity staining of Ad-β-gal-transduced VSMC (right panel) illustrates high transduction rate and expression of adenovirus in VSMC cultures. D. Representative ploidy profiles from transduced VSMC cultures (adenovirus is indicated in figure) show increased polyploidization of Ad-Nox4 cultures in comparison to controls, Ad-β-gal and dominant negative Ad-Nox4-ΔFAD 10 days post transduction. E. Table showing percent of cells in 2N, 4N and 8N ploidy classes as well as a ploidy index for VSMC cultures transduced with Ad-β-gal and Ad-Nox4. DNA content was determined by FACS analysis and ploidy classes are represented as a fraction of the total cell population. The polyploidy index was calculated by the formula ((% 4N + 8N)/% 2N). Asterisk (*) indicates a significant difference (p<0.005) as determined using the student’s t-test.

Figure 4. Nox4 overexpression downregulates survivin in VSMC cultures

Primary cultures of VSMC transduced with indicated adenovirus were harvested for western blot analysis to detect levels of Nox4 and survivin. Bands have been quantitated, as in Figure 1, showing fold changes of 2-3 fold upregulation for Nox4 and a 2-3 fold downregulation for survivin. GAPDH and β-actin were used as loading controls. These results are representative of 4 experiments.

Figure 5. Nox4 overexpression downregulates survivin mRNA

Primary cultures of VSMC were transduced with Ad-β-gal or Ad-Nox4 and RNA was harvested 10 days post transduction. Real time PCR analysis indicates that elevated Nox4 expression downregulates survivin mRNA levels in comparison to control. The above graph represents the average of three independent experiments. Statistical significance (p = 0.0332) was determined using the student’s t-test.