Short‐Term Statin Treatment Reduces, and Long‐Term Statin Treatment Abolishes, Chronic Vascular Injury by Radiation Therapy

Abstract

Background

The incidental use of statins during radiation therapy has been associated with a reduced long‐term risk of developing atherosclerotic cardiovascular disease. We examined whether irradiation causes chronic vascular injury and whether short‐term administration of statins during and after irradiation is sufficient to prevent chronic injury compared with long‐term administration.

Methods and Results

C57Bl/6 mice were pretreated with pravastatin for 72 hours and then exposed to 12 Gy X‐ray head‐and‐neck irradiation. Pravastatin was then administered either for an additional 24 hours or for 1 year. Carotid arteries were tested for vascular reactivity, altered gene expression, and collagen deposition 1 year after irradiation. Treatment with pravastatin for 24 hours after irradiation reduced the loss of endothelium‐dependent vasorelaxation and protected against enhanced vasoconstriction. Expression of markers associated with inflammation (NFκB p65 [phospho‐nuclear factor kappa B p65] and TNF‐α [tumor necrosis factor alpha]) and with oxidative stress (NADPH oxidases 2 and 4) were lowered and subunits of the voltage and Ca²⁺ activated K⁺ BK channel (potassium calcium‐activated channel subfamily M alpha 1 and potassium calcium‐activated channel subfamily M regulatory beta subunit 1) in the carotid artery were modulated. Treatment with pravastatin for 1 year after irradiation completely reversed irradiation‐induced changes.

Conclusions

Short‐term administration of pravastatin is sufficient to reduce chronic vascular injury at 1 year after irradiation. Long‐term administration eliminates the effects of irradiation. These findings suggest that a prospective treatment strategy involving statins could be effective in patients undergoing radiation therapy. The optimal duration of treatment in humans has yet to be determined.

Nonstandard Abbreviations and Acronyms

BK channel

Ca²⁺ activated K⁺ channel

eNOS

endothelial nitric oxide synthase I

ICAM1

intercellular adhesion molecule 1

KCNM

potassium calcium‐activated channel subfamily M

Research Perspective.

What Is New?

• In mice that underwent radiation to head and neck, treatment with pravastatin for only 24 hours after irradiation reduced the loss of endothelium‐dependent vasorelaxation, protected against enhanced vasoconstriction and expression of markers associated with inflammation, oxidative stress and collagen deposition, and normalized BK channel subunit expression.

• The findings of this study in mice suggest that statins may alleviate or prevent the reported accelerated progression of carotid disease and increased stroke risk in patients with head and neck cancer treated with radiation therapy.

• Treatment with pravastatin for 1 year after irradiation completely reversed irradiation‐induced changes, including increased collagen expression.

What Question Should Be Addressed Next?

• A prospective study of statins before and during radiation therapy may be warranted to determine the optimal duration of treatment in patients with head and neck cancer.

Radiation therapy has been associated with an elevated risk of developing vascular disease. ¹ Following radiation therapy for head‐and‐neck cancer, the risk for carotid stenosis is strongly increased ² , ³ , ⁴ , ⁵ because the carotid artery is adjacent to the targeted lymphatic structures and usually included in the radiation field. In patients who had undergone radiation therapy for head and neck cancer, the rate of progression of carotid artery stenosis to >50% was 3‐fold higher than in patients who had not received radiation therapy but were matched for the severity of carotid artery stenosis at baseline. ⁶

Although to date no drugs have been designed specifically to prevent irradiation‐induced vascular disease, the incidental use of statins at the time of, or after, radiation therapy for head and neck cancer has been associated with a lower risk of stroke. ⁷ , ⁸ In spite of these promising observations, prospective clinical trials directly testing whether statin treatment reduces vascular disease in this population have not been undertaken, in part because of the long interval between radiation therapy and clinical events. ⁷ Given these considerations, studies in preclinical models can provide valuable information.

We recently reported that in C57Bl/6 mice, treatment with pravastatin for up to 10 days after irradiation prevented endothelial dysfunction and mitochondrial damage. ⁹ In the current study, we sought to determine whether irradiation induces chronic injury by upregulating mediators of inflammation and oxidative stress, as reported in humans, ¹⁰ and whether statin therapy protects from chronic vascular injury. Additionally, we tested 2 regimens of pravastatin administration to determine the duration of therapy necessary to prevent vascular injury. Specifically, we looked at a 1‐day treatment after irradiation (short‐term) and continuous 1‐year treatment after irradiation (chronic, long‐term). Irradiation acutely induces oxidative stress, and statins like pravastatin have known antioxidants effects. ¹¹ , ¹² Therefore, we aimed to investigate whether a brief course of statin treatment could confer protective benefits. Given the increased risk for carotid artery stenosis in patients with head and neck cancer after of radiation therapy, coupled with the established role of statins in mitigating carotid disease progression and stroke risk, ¹³ , ¹⁴ we opted to include continuous treatment in analogy to chronic treatment with statins in human patients. We examined the effects on dilation and constriction of the common carotid artery, the expression of markers of inflammatory and oxidative stress, and of structural components of the vascular wall.

METHODS

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Mice

All experimental procedures were approved by the Institutional Animal Care and Use Committees of both the University of Iowa and the Iowa City VA Health Care System and complied using the standards of the Institute of Laboratory Animal Resources, National Academy of Sciences. Male and female C57BL/6J mice were obtained from Jackson Laboratories (#000664). Mice were housed at 23 °C on a 12:12‐hour light/dark cycle and had free access to chow and water. All mice were 12 to 16 weeks of age at the time of treatment.

Statin Treatment

Six treatment groups were included: (1) Vehicle nonirradiated: this group received vehicle (filtered tap water) and was sham irradiated (N=6); (2) Vehicle irradiated: this group received vehicle (filtered tap water) and underwent head‐and‐neck irradiation (12 Gy X‐ray) (N=6); (3) ST‐Prava nonirradiated: the short‐term pravastatin control group was administered pravastatin in drinking water beginning 3 days before and continuing for 1 additional day after sham treatment (nonirradiated) (N=6); (4) ST‐Prava irradiated: the short‐term pravastatin irradiated group was administered pravastatin in drinking water beginning 3 days before irradiation and continuing for 1 additional day after irradiation (N=6); (5) LT‐Prava nonirradiated: this long‐term pravastatin control group received pravastatin in drinking water from 3 days before sham irradiation and maintained through 12 months (N=6); and (6) LT‐Prava irradiated: the long‐term pravastatin irradiated group received pravastatin in drinking water from 72 hours before irradiation and maintained through 12 months after (N=6).

Pravastatin was given orally in drinking water, which was provided ad libitum, resulting in a dose of approximately 30 mg/kg per day. ¹⁵ There were 6 animals per group and each animal was considered an experimental unit. No animals were excluded from the analysis. All animals in 1 cage received the same treatment provided in the drinking water. The primary outcome measure was change in vasoreactivity and mRNA expression. The experimenter was blinded to the treatment. As for the studies on mRNA expression and collagen deposition, the experiment and analysis were performed by different scientists.

Irradiation

Mice were anesthetized with isoflurane and irradiated using the XStrahl Small Animal Radiation Research Platform with a single anterior–posterior beam and a beam quality of 0.67 mm Cu, as previously described. ⁹ Sham‐treated mice were anesthetized but did not undergo irradiation.

The dose rate was 3.6 Gy/min and calibrated at 2 cm depth in water, in accordance with the AAPM TG‐61 protocol. A dose of 12 Gy X‐ray, which equates to EQD2 dose of 36 Gy (α/β of 3), was delivered to the head and neck in a single session. Simulation was performed by computed tomography. The accuracy of dosimetry by the Small Animal Radiation Research Platform was ensured by quarterly measurements of the ion chamber by a medical physicist. ¹⁶

Vascular Reactivity

Arterial rings were prepared from the common carotid and second‐branch mesenteric resistance arteries. Isometric tension was measured after mounting the rings in a small vessel dual chamber myograph. Following equilibration in Krebs solution bubbled with CO₂ at 37 °C and at pH 7.4 for 30 minutes, the rings were stretched to their optimal physiological lumen diameter for 1 hour to develop active tension. The rings were then preconstricted with phenylephrine (3×10⁻⁵ mol/L), after which they were treated with acetylcholine (10⁻⁸ to 3×10⁻⁵ mol/L) or sodium nitroprusside (10⁻⁸ to 3×10⁻⁵ mol/L) to generate cumulative concentration‐response curves.

Mesenteric resistance arteries served as control arteries from a vascular bed outside the radiation field and were treated in the same fashion. Data from male and female mice were initially analyzed separately and then combined because no differences were seen.

Quantitative Real‐Time Polymerase Chain Reaction

Total RNA extracted from carotid artery lysates was reverse transcribed and amplified in a ViiA 7 Real‐Time PCR System (Applied Biosystems, Foster City, CA). Primers were designed by Integrated DNA Technologies (Coralville, IA) to amplify the following genes: phospho‐nuclear factor kappa B p65, tumor necrosis factor alpha, intercellular adhesion molecule 1, endothelial nitric oxide synthase (eNOS), NADPH oxidases 2 and 4, myosin heavy chain 11, myosin light‐chain kinase, smooth muscle actin, collagen type I α2 chain, potassium calcium‐ activated channel subfamily M alpha 1 (KCNMA1), potassium calcium‐activated channel subfamily M regulatory beta subunit 1 (KCNMB1), and ribosomal 18S or β‐actin (internal control). Primer sequences are provided in Table S1.

Masson Trichrome Staining

Carotid arteries were fixed in 4% paraformaldehyde, embedded in OCT compound, and sliced to 10 μm sections. These samples were stained with Masson's trichrome reagent for evaluation of collagen deposition.

Statistical Analysis

Data were expressed as mean±SEM and analyzed using GraphPad Prism 9.0 software. All data sets were analyzed for normality and equal variance. Kruskal–Wallis and Dunn's post hoc tests were used for data sets when a normal distribution could not be assumed. Comparisons were made to vehicle‐irradiated conditions. One‐way ANOVA, followed by Tukey's multiple comparison test, was used for data sets with a normal distribution. Two‐way ANOVA followed by Tukey's multiple comparison test was used for grouped data sets. A P value <0.05 was considered significant.

RESULTS

Short‐Term Treatment With Pravastatin Partially Alleviates Abnormal Vascular Reactivity at 1 Year Following Irradiation, Whereas Long‐Term Treatment Prevents It

At 1 year after irradiation, we measured the dilation and constriction of the common carotid artery in response to different treatments: acetylcholine for endothelium‐dependent dilation, sodium nitroprusside for endothelium‐independent dilation, and phenylephrine for vasoconstriction.

At this time point, we observed that relaxation in response to acetylcholine was significantly reduced in the common carotid arteries of irradiated mice compared with nonirradiated sham control mice (Figure 1A through 1C). In mice that had received ST‐Prava treatment (1 day), endothelium‐dependent relaxation was impaired compared with nonirradiated mice and improved compared with irradiated mice that had not received pravastatin. (Figure 1B and 1D). However, irradiated mice that underwent LT‐Prava treatment (1 year) exhibited the same level of endothelium‐dependent relaxation than mice in the nonirradiated group, indicating complete preservation (Figure 1B and 1E). Similar results were observed in both males and females (Figures S1 and S2). Control experiments assessing the effects of irradiation and statins on dilation by sodium nitroprusside confirmed that neither affected endothelium‐independent carotid relaxation (Figure 1F). Moreover, in mesenteric resistance arteries outside the radiation field, dilation in response to acetylcholine was not affected by either irradiation or statin treatment (Figure 1G).

Figure 1. Pravastatin treatment preserves endothelial function ex vivo following head and neck irradiation.

A, Schematic of the study design. Control mice were treated with Vehicle, underwent head‐and‐neck irradiation (12 Gy X‐ray) or sham treatment (nIR). Vascular reactivity was tested at 1‐year after IR or nIR. For short‐term (1‐day) pravastatin treatment, pravastatin (30 mg/kg per day) was started at 72 hours before and continued for 1 day after IR or sham treatment. For long‐term (1‐year) pravastatin treatment, pravastatin (30 mg/kg per day) was started at 72 hours before and continued for 1 year after IR or sham treatment. Vascular reactivity was tested at 1 year after IR. B, Effects of ST‐Prava, LT‐Prava or vehicle treatment on endothelium‐dependent relaxation of the common carotid artery in response to acetylcholine at 1 year after head‐and‐neck irradiation (IR, 12 Gy X‐ray) or sham treatment (nIR). C, Vehicle treatment, data as in (B). D, ST‐Prava, data as in (B), E, LT‐Prava, data as in (B). F, Effects of ST‐Prava, LT‐Prava, or vehicle treatment on endothelium‐independent relaxation of the common carotid artery in response to sodium nitroprusside at 1 year after head‐and‐neck irradiation (IR, 12 Gy X‐ray) or sham treatment (nIR). G, Effects of ST‐Prava, LT‐Prava, or vehicle treatment on endothelium‐dependent relaxation of mesenteric resistance arteries at 1 year after head‐and‐neck irradiation (IR, 12 Gy X‐ray) or sham treatment (nIR). N=6 mice per group. P values were determined using repeated measures 2‐way ANOVA followed by Tukey's post hoc test and are shown for 10⁻⁴ mol/L Ach indicates acetylcholine; IR, irradiated; LT‐Prava, long‐term pravastatin; MRA, mesenteric resistance arteries; nIR, nonirradiated; SNP, sodium nitroprusside; and ST‐Prava, short‐term pravastatin.

Vasoconstriction in response to phenylephrine was increased in the irradiated group compared with the nonirradiated group (Figure 2A through 2C), and both ST‐Prava and LT‐Prava treatment normalized the vasoconstriction after irradiation (Figure 2B, 2D, 2E). Similar results were observed from both males and females (Figures S1 and S2). In mesenteric resistance arteries outside the radiation field, constriction in response to phenylephrine was not altered by either irradiation or statin treatment (Figure 2F).

Figure 2. Pravastatin treatment prevents enhanced carotid artery constriction ex vivo following head and neck irradiation.

A, Schematic of the study design. Control mice were treated with Vehicle, underwent head‐and‐neck irradiation (IR, 12 Gy X‐ray) or sham treatment (nIR). Vascular reactivity was tested at 1‐year after IR or nIR. For short‐term (1‐day) pravastatin treatment, pravastatin (30 mg/kg per day) was started at 72 hours before and continued for 1 day after IR or sham treatment. For long‐term (1‐year) pravastatin treatment, pravastatin (30 mg/kg per day) was started at 72 hours before and continued for 1 year after IR or sham treatment. Vascular reactivity was tested at 1 year after IR. B, Effects of ST‐Prava, LT‐Prava, or vehicle treatment on constriction of the common carotid artery in response to phenylephrine at 1 year after head‐and‐neck irradiation (IR, 12 Gy X‐ray) or sham treatment (nIR), in mice treated as in Figure 1. C, Vehicle treatment, data as in (B). D, ST‐Prava, data as in (B), E, LT‐Prava, data as in (B). F, Effects of ST‐Prava, LT‐Prava, or vehicle treatment on constriction mesenteric resistance arteries at 1 year after head‐and‐neck irradiation (IR, 12 Gy X‐ray) or sham treatment (nIR). N=6 mice per group. P values were determined using repeated measures 2‐way ANOVA followed by Tukey's post hoc test and are shown for 10⁻⁴ mol/L IR indicates irradiated; LT‐Prava, long‐term pravastatin; MRA, mesenteric resistance arteries; nIR, nonirradiated; PE, phenylephrine; and ST‐Prava, short‐term pravastatin.

Short‐Term Treatment With Pravastatin Reduces Changes in the Expression of Markers of Inflammation, Oxidative Stress, and Structural Wall Remodeling at 1 Year After IR, Whereas Long‐Term Treatment Abolishes Them

To identify the protective effects of short‐ versus long‐term treatment with pravastatin, we used quantitative real‐time polymerase chain reaction to analyze the expression of markers associated with inflammation, oxidative stress, and structural remodeling. At 1 year after irradiation, carotid arteries from mice treated with vehicle and irradiation exhibited higher levels of mRNAs encoding phospho‐nuclear factor kappa B p65, tumor necrosis factor alpha, and intercellular adhesion molecule 1 compared with vehicle‐treated nonirradiated mice. However, these effects were abolished by LT‐Prava treatment and mildly reduced by ST‐Prava treatment (Figure 3A through 3C). In contrast, mRNA for eNOS, which regulates nitric oxide synthesis, was significantly lower in carotid arteries from irradiated mice compared with nonirradiated mice (Figure 3D). Both pravastatin treatments prevented this difference, with the long‐term treatment showing a more significant effect. Additionally, the expression of NADPH oxidase 2 and 4 mRNAs, which encode NADPH oxidases expressed in the vascular wall, was higher in carotid arteries from irradiated mice compared with nonirradiated vehicle‐treated mice (Figure 3E and 3F). LT‐Prava treatment blocked the effect of both NADPH oxidase 2 and 4, whereas ST‐Prava treatment specifically blocked the effect of NADPH oxidase 4.

Figure 3. Pravastatin reduces IR‐induced expression of genes in inflammatory oxidative stress pathways.

All panels compare gene expression in common carotid arteries of C57BL/6J mice pretreated with vehicle or pravastatin for 72 hours, subjected to sham treatment (nIR) or IR (12 Gy), followed by short‐term (1‐day) or long‐term (1 year) pravastatin treatment. Quantitative RT‐PCR was performed at 1 year after IR. Quantitative RT‐PCR for NFκB‐p65 (A), TNFα (B), ICAM1 (C), eNOS (D), NOX2 (E), and NOX4 (F). n=6 mice per group for A through F. P values were determined using Kruskal–Wallis test. eNOS indicates endothelial nitric oxide synthase; ICAM1, intercellular adhesion molecule 1; IR, irradiated; LT‐Prava, long‐term pravastatin; NOX2, NADPH oxidase 2; NOX4, NADPH oxidase 4; NFκB‐p65, phospho‐nuclear factor kappa B p65; nIR, nonirradiated; RT‐PCR, real‐time polymerase chain reaction; ST‐Prava, short‐term pravastatin; and TNFα, tumor necrosis factor alpha.

Furthermore, irradiation reduced the levels of mRNAs encoding smooth muscle genes, including myosin heavy chain 11, myosin light‐chain kinase, and smooth muscle actin (Figure 4A through 4C). Only LT‐Prava treatment abolished this effect. Findings were for SM22α (Figure 4D).

Figure 4. Pravastatin normalizes IR‐induced expression of genes of smooth muscle cell structural genes and ion channels implicated in enhanced constriction after IR.

All panels compare gene expression in common carotid arteries of C57BL/6J mice pretreated with vehicle or pravastatin for 72 hours, subjected to sham treatment (nIR) and IR (12 Gy), followed by short‐term (1‐day) or long‐term (1 year) pravastatin treatment. Quantitative RT‐PCR was performed at 1 year after IR. Quantitative RT‐PCR for myosin heavy chain 11 (A), myosin light‐chain kinase (B), smooth muscle actin (C), SM22α (D), potassium calcium‐activated channel subfamily M alpha 1 (E), and potassium calcium‐activated channel subfamily M regulatory beta subunit 1 (F). N=6 mice per group for A through D, n=4 for E and F. P values were determined using the Kruskal–Wallis test. IR indicates irradiated; KCNMA1, potassium calcium‐activated channel subfamily M alpha 1; KCNMB1, potassium calcium‐activated channel subfamily M regulatory beta subunit 1; LT‐Prava, long‐term pravastatin; MLCK, myosin light‐chain kinase; MYH11, myosin heavy chain 11; nIR, nonirradiated; RT‐PCR, real‐time polymerase chain reaction; SMA, smooth muscle actin; and ST‐Prava, short‐term pravastatin.

Given that vasoconstriction in response to phenylephrine was increased in the irradiated group compared with the nonirradiated group and enhanced vasoconstriction normalized with both ST‐Prava and LT‐Prava treatment, we investigated whether the expression of subunits of the voltage and Ca²⁺ activated K⁺ (BK) channel is altered under these conditions. Previous studies suggested that altered posttranscriptional regulation and decreased mRNA levels of BK channels in smooth muscle cells contribute to increased constriction after irradiation. ¹³ , ¹⁴ , ¹⁵ , ¹⁶ Here, we detected significant increases in mRNA levels of the BK channel subunits KCNMA1 and KCNMB1 with irradiation. Both LT‐Prava and ST‐Prava treatment abolished this effect (Figure 4E and 4F).

Finally, we assessed the impact of pravastatin treatment on collagen expression induced by irradiation. Quantitative real‐time polymerase chain reaction showed that expression of the collagen type I α1 and collagen type I α2 chains was elevated after irradiation in vehicle‐treated mice, and LT‐Prava treatment abolished this effect with a moderate effect by ST‐Prava treatment (Figure 5A and 5B). In accordance, increased collagen deposition was seen in carotid arteries of irradiated mice by Masson's trichrome staining compared with vehicle‐treated mice (Figure 5C and 5D).

Figure 5. Pravastatin reduces IR‐induced collagen deposition.

A and B, All panels compare gene expression in common carotid arteries of C57BL/6J mice pretreated with vehicle or pravastatin for 72 hours, subjected to sham treatment (nIR) or IR (12 Gy), followed by short‐term (1‐day) or long‐term (1 year) pravastatin treatment. Quantitative RT‐PCR for collagen type I alpha 1 chain (A) and collagen type I alpha 2 chain (B). C, Masson's trichrome staining of sections of the common carotid artery of C57BL/6J mice pretreated with vehicle or pravastatin for 72 hours, subjected to a sham treatment (nIR) or IR (12 Gy), then short‐term (1‐day) or long‐term (1 year) pravastatin treatment. Scale bar=10 μm, arrows point to collagen deposition. D, Quantification of collagen deposition as in C. N=6 mice per group for A and B, n=4 for C and D. P values were determined using Kruskal–Wallis test. COL1A1 indicates collagen type I alpha 1 chain; COL1A2, collagen type I alpha 2; IR, irradiated; LT‐Prava, long‐term pravastatin; nIR, nonirradiated; RT‐PCR, real‐time polymerase chain reaction; and ST‐Prava, short‐term pravastatin

DISCUSSION

In this study, we aimed to determine whether radiation‐induced vascular injury resulting in changes in vascular reactivity and expression of markers related to inflammation and oxidative stress could be detected 1 year after irradiation. Additionally, we investigated the protective effects of treatment with pravastatin, either for 1 day only or for 1 year after irradiation, against vascular injury. Our study yielded 3 significant findings. First, we observed impaired endothelium‐dependent dilation and increased constriction in the common carotid artery 1 year after irradiation. Second, there was an elevation in the expression of mRNAs encoding inflammatory markers, NADPH oxidase 2, and NADPH oxidase 4 and BK channel subunits KCNMA1 and KCNMB1, whereas mRNAs encoding eNOS and smooth muscle structural proteins showed lower expression in irradiated mice compared with nonirradiated mice. Third, LT‐Prava treatment throughout the 1‐year post irradiation period eliminated the effects of irradiation, whereas even a 1‐day short‐term treatment normalized constriction and the expression of NADPH oxidase 4, KCNMA1, and KCNMB1, with some minor effects on endothelium‐dependent dilation and the expression of other proinflammatory proteins and collagen mRNA.

In patients with head and neck cancer, irradiation drives the progression of carotid arterial disease, leading to carotid artery stenosis and stroke. ¹⁷ However, there are currently no specific therapies available to treat irradiation‐induced cardiovascular disease. As a result, preventive treatments such as aspirin, colchicine, and statins have been proposed instead. ¹⁸ Interestingly, 1 study reported that the incidental use of statins lowered the risk of stroke in patients after irradiation for head and neck cancer. ⁷ However, in adult survivors of childhood cancer, atorvastatin did not improve endothelial function or arterial stiffness, possibly because the atherosclerotic risk in this population is lower than in adult cancer patients. ¹⁹ As for the underlying mechanism for accelerated disease progression, studies in humans have identified changes in the expression of intercellular adhesion molecule 1, vascular cell adhesion protein 1, E‐ and P‐selectin, eNOS, and increased NF‐κB signaling within a span of up to 10 years after irradiation. ¹⁰ , ²⁰ , ²¹ Preclinical studies in mice are expected to provide additional insights because the same mechanisms of arterial inflammation and oxidative stress have been implicated in experimental irradiation‐related vascular disease. However, in preclinical models, altered vascular reactivity, inflammation, and oxidative stress have only been studied within a few weeks after irradiation. ²² , ²³ , ²⁴ , ²⁵ Our experiments, conducted 1 year after irradiation, aimed to bridge the knowledge gap regarding long‐term effects in preclinical models. Our findings confirm the presence of inflammation with expression of proinflammatory genes, likely driven by NF‐κB and oxidative stress at this time point. These changes were reduced by statin treatment. Irradiation impairs endothelial‐dependent vasodilation through NO‐dependent pathways in humans ²⁶ , ²⁷ and in preclinical models. ²⁸ , ²⁹ Endothelial dysfunction is a strong predictor of atherosclerotic risk and adverse cardiovascular events, including stroke. ³⁰ , ³¹ Statin therapy maintains NO production by preserving expression of eNOS mRNA. ³² Statin therapy also protects against uncoupling of eNOS, thereby decreasing reactive oxygen species production. Our findings suggest that statin therapy, by reducing the expression of NOX proteins, ³³ lowers reactive oxygen species production and increases eNOS levels, resulting in preserved vasodilation even at 1 year after irradiation.

Although less is known about the effects of irradiation on vasoconstriction compared with vasodilation, previous studies indicate that altered posttranscriptional regulation and decreased mRNA transcription of BK channel subunits in smooth muscle cells may drive increased constriction. ³⁴ , ³⁵ , ³⁶ , ³⁷ Opposite to our predictions, mRNA levels of the BK channel subunits KCNMA1 and KCNMB1 were higher at 1 year after irradiation compared with control nonirradiated conditions. Both short‐ and long‐term statin treatments normalized mRNA expression. Our findings warrant further studies of BK channel protein levels in smooth muscle cells of the vascular wall after irradiation. This will be critical because the conclusions in previous studies were solely drawn from electrophysiological studies with BK and PKC (protein kinase C) inhibitors and quantification of BK channel subunit mRNA transcripts. Moreover, to get a complete picture, there is a need to study other channels relevant for vasoconstriction, including voltage‐gated K+, ATP‐sensitive potassium, and L‐type voltage‐dependent Ca(2+) channels. Increased fibrosis is a well‐established characteristic of pathology in normal tissue after irradiation. ³⁸ , ³⁹ In our studies, pravastatin reduced collagen levels, which may also have contributed to the normalization of vasoreactivity by this treatment.

In this study, we sought to address the lack of mechanistic data on vascular dysfunction after irradiation. Additionally, preclinical models have not been used to explore various preventive treatments. Our findings demonstrate that treatment with statins during and after irradiation can mitigate the adverse effects on vascular function and atherosclerotic risk factors. Specifically, administering pravastatin for just 1 day post irradiation improved dilation, normalized constriction, and inhibited the increased expression of NADPH oxidase 4 and BK channel subunit KCNMB1. Moreover, it attenuated changes in mRNA levels of other markers induced by irradiation. These results suggest that a short course of statin treatment may effectively alleviate aspects of vascular injury following irradiation.

Our study has several limitations. First, we opted to perform all experiments in the same samples. Consequently, the current data set is limited to mRNA expression and vasoreactivity due to the small sample quantity. Additional experiments to ascertain protein levels of inflammatory and structural markers are warranted. Second, further studies will be essential to determine the ideal duration of statin treatment and to fully establish the molecular mechanisms that contribute to enhanced constriction after irradiation. Third, for this study, we tested only pravastatin, a moderate‐intensity stating that is not the preferred choice as a cholesterol‐lowering drug. The selection of pravastatin for this study was dictated by both our prior research and existing literature, on the protective effects of pravastatin on irradiation‐induced skin injury in patients with head and neck cancer ⁴⁰ and in other normal tissues such as gut. ¹⁵ , ⁴¹ Moreover, our recent research emphasizes the importance of choosing the right statin in the context of irradiation. Mitochondrial dysfunction has been suggested as a key factor in the development of cardiovascular disease induced by irradiation. ⁴² Certain statins, such as atorvastatin, might exacerbate mitochondrial damage. ⁴³ , ⁴⁴ We recently demonstrated that atorvastatin in contrast to pravastatin contributed to mitochondrial damage that persisted for 10 days following irradiation. ⁹ Further studies are needed to understand the class effects of statins in irradiation injury. Lastly, it will be important to establish how statin treatment affects irradiation‐induced atherosclerosis formation in mouse models.

Conclusions

In conclusion, this study demonstrates the long‐term vascular effects of irradiation‐induced injury and the protective potential of pravastatin treatment in mice. Impaired endothelial function and altered gene expression patterns were observed 1 year post irradiation, indicative of vascular dysfunction. Both short‐term and pravastatin treatment alleviated and long‐term pravastatin treatment reversed these effects, suggesting its efficacy in mitigating radiation‐induced vascular injury. These findings underscore the importance of statin therapy in managing cardiovascular complications following radiation therapy for head and neck cancer. Further research is warranted to optimize statin treatment strategies and elucidate underlying molecular mechanisms.

Sources of Funding

This project was supported by grants from the National Institutes of Health (R01 EY031544 to Isabella M. Grumbach); the American Heart Association (18IPA 34170 003 to Isabella M. Grumbach and 2021CDA 853 499 to Karima Ait‐Aissa); and the US Department of Veterans Affairs (I01 BX000163).

Disclosures

None.

Supporting information

Table S1

Figures S1–S2