Calcium channel blockers increase the risk of aortic aneurysm and dissection

Domain: physics
Published: 2025-12-25
Editorial outcome: minor_revision

Abstract
Aortic aneurysm and dissection (AAD) are life-threatening conditions without effective medications. Impaired contractility of vascular smooth muscle cells (VSMCs) is strongly linked to AAD, but the role of calcium channel blockers (CCBs), which directly inhibits VSMC contractility, in AAD remains unclear. Here we showed data from 501,878 initially AAD-free participants in UK Biobank. Over a median follow-up of 13.5 years, CCB users had higher AAD risk (HR=1.31) than hypertensive patients not receiving antihypertensive treatment. In mouse models of AAD, CCBs significantly aggravated aortic stiffness and AAD development. For patients with type B aortic dissection who underwent endovascular repair, CCBs limited AAD regression compared with other antihypertensives. Moreover, silencing of protein kinase cGMP-dependent 1 (PRKG1) significantly mitigated CCB-aggravated AAD progression. These findings suggest that CCBs may increase AAD risk and post-stent surgery prognosis, highlighting the need for caution when prescribing CCBs to hypertensive patients at risk for AAD.

Introduction
Aortic aneurysm and dissection (AAD) are life-threatening cardiovascular conditions associated with high mortality rates. Aortic aneurysm refers to the irreversible enlargement of the aorta, resulting in a diameter exceeding 1.5 times that of adjacent normal vessels. Aortic dissection is defined as disruption of the intima leading to separation of the aortic wall layers followed by the formation of a false lumen1. Depending on the location, AAD are classified into thoracic aortic aneurysm/dissection (TAAD) and abdominal aortic aneurysm (AAA)2,3. Although recognized as distinct disease entities, TAAD and AAA share common pathological changes, including vascular extracellular matrix degradation, inflammation, and dysfunction of vascular smooth muscle cells (VSMCs). Currently, the management of AAD relies solely on surgical intervention, including artificial blood vessel replacement and endovascular stent graft repair4. However, at present, only β-blockers or angiotensin II receptor blockers (ARBs) are recommended for aortic aneurysms in patients with Marfan syndrome. There are currently no proven drugs to prevent the occurrence of or slow the expansion of the aorta in patients with non-hereditary AAD, including blood pressure-lowering drugs5–7, lipid-modifying medications8, anti-inflammatory or antibiotics drugs9–11, anti-thrombotic drugs12.

Hypertension has long been recognized as an independent risk factor for AAD13,14. In clinical practice, antihypertensive drugs, including angiotensin II receptor blockers (ARBs), calcium channel blockers (CCBs), and β-blockers, are commonly prescribed to AAD patients to reduce pressure on the aortic wall, but the effect of various antihypertensive drugs on AAD patients remains unclear. Notably, emerging evidence indicates that the development of AAD is attributed to the impaired VSMC contractility15. Reduced expression of contractile apparatus proteins (e.g., myosin heavy chain [MYH11], smooth muscle α-actin [SM α-actin], and smooth muscle protein 22-α [SM22-α]) is observed in the aortas of patients with TAAD and AAA16. Additionally, genetic mutations in myosin heavy chain 11 (MYH11), actin α 2 (ACTA2), myosin light chain kinase (MYLK), and cGMP-dependent protein kinase type I (PRKG1) are linked to TAAD17,18. Moreover, patients with AAD have shown reduced vascular contractility, accompanied by changes in mechanical properties, such as increased stiffness19–21. This raises concerns about whether antihypertensive drugs that inhibit VSMC contractility, such as CCBs22, pose potential risks to AAD onset. Previous studies have reported that CCBs (amlodipine, diltiazem, and azelnidipine), at doses 5–10 times higher than clinical doses, may alleviate angiotensin II (Ang II)-induced AAD formation when co-administered with Ang II in mice23–26. However, a single study revealed that amlodipine and verapamil promoted aorta expansion in mice with Marfan syndrome (MFS)27. Furthermore, in a randomized controlled trial involving 224 patients with small AAAs, amlodipine failed to delay aortic expansion7. Currently, however, all clinical studies have focused on the medication treatment of patients with diagnosed AAD, while the impact of CCB use in hypertensive patients on the incidence of AAD remains unclear.

In this study, our objective was to evaluate the impact of CCBs on the risk of AAD. Here we report results of (1) the association between CCBs and their subtypes with subsequent AAD occurrence, using data from the UK Biobank; and (2) the effects of CCBs on both murine models and post-stent AAD patients.

Results
Association of CCB or subtype use with the risk of AAD in UKB participants
We first analyzed the association between CCBs and the incidence of AAD using data from the UK Biobank (Fig. 1a). Among the 501,878 participants initially free of AAD, 28.3% were identified as having hypertension (Supplementary Table 1). These hypertensive participants were divided into three groups based on their medication use: 8.7% were not taking any antihypertensives, and 6.8% were using CCBs, either exclusively (5.7%) or in combination with other antihypertensive medications (1.1%). The remaining 12.8% were classified as users of antihypertensive medications other than CCBs (Supplementary Tables 2, 3). The baseline characteristics of the participants are shown in Supplementary Table 4. Hypertensive participants, regardless of medication use, were generally older, had higher body mass index (BMI), and higher systolic blood pressure (SBP) than those without hypertension. Among hypertensive groups, CCB users were older, had more men, fewer white participants, and lower total cholesterol levels than untreated individuals or those using other antihypertensive medications.

During a median follow-up period of 13.5 years, 4,327 cases of AAD were identified, including 1520 TAAD and 2580 AAA (Fig. 1a and Supplementary Table 5). As expected, participants without hypertension had significantly lower risks of AAD, TAAD, and AAA compared to those with hypertension but without antihypertensive medication (P<0.001 for all) (Fig. 1b). We further found that participants with hypertension and concurrent CCB use had greater risks of AAD (adjusted HR=1.31, 95% CI: 1.17–1.48), TAAD (adjusted HR=1.23, 95% CI: 1.00–1.50), and AAA (adjusted HR=1.32, 95% CI: 1.13–1.53) than hypertensive patients not receiving antihypertensive medication (Fig. 1b). To minimize the indication bias that medication use itself might reflect disease severity, we restricted the analyses to the participants receiving antihypertensive medications and observed that CCB users still had a significantly greater risk of AAD (HR=1.23, 95% CI: 1.09–1.38) than other antihypertensive medication users, with consistent results in both males and females and a relatively greater effect observed in females (Fig. 1c and Supplementary Tables 6, 7). Furthermore, among the participants with prevalent AAD, CCB use was associated with increased mortality (HR=1.52, 95% CI: 1.04–2.21) (Supplementary Table 8).

CCBs are generally divided into three main subtypes based on their chemical structure and action mechanisms: dihydropyridines, benzothiazepines and phenylalkylamines28 (Fig. 1d). All subtypes of CCBs were associated with a significantly greater AAD risk than no CCB use among participants with hypertension, with phenylalkylamine CCBs presenting the highest risk (HR=2.33, 95% CI: 1.58–3.45), benzothiazepine CCBs showing an intermediate risk (HR=2.07, 95% CI: 1.65–2.60), and dihydropyridine CCBs having a relatively lower risk (HR=1.29, 95% CI: 1.14–1.46), after further adjusting for concurrent use of other blood pressure medications (Fig. 1e). Sensitivity analysis considering the competing risk of death, using a broader definition of hypertension, excluding participants with coronary artery diseases, adjusting for eGFR, or removing participants with hypertension who used both CCBs and other antihypertensive medications yielded similar results, supporting the robustness of our study (Supplementary Tables 9–13).

CCBs aggravated both thoracic and abdominal aortic aneurysm formation in AngII-infused mouse model
We next investigated the causal effect of different CCB subtypes on the development of aortic aneurysms in an angiotensin II (AngII)-infused mouse model (Fig. 2a). Sixteen-week-old C57BL/6J mice were subcutaneously infused with AngII (1000 ng/kg·min−1) via minipumps for a total of 28 days. First, 70 sixteen-week-old male C57BL/6J mice were initially infused with AngII for 7 days to induce hypertension. During this period, 11 mice either died of aneurysm rupture or were diagnosed with aortic aneurysm via ultrasound. Then, the remaining 59 hypertensive mice were randomly assigned to receive either a vehicle or one of four CCBs: amlodipine (1 mg/kg/day), nifedipine (20 mg/kg/day), diltiazem (8 mg/kg/day) or verapamil (12 mg/kg/day) (Supplementary Fig. 1a). Saline-infused mice were treated with vehicle and served as a negative control. As expected, all four CCB treatments significantly reduced SBP in the AngII-infused mice (Fig. 2b).

During CCB administration, we performed weekly ultrasound to monitor the diameters of the thoracic aorta (aortic root, ascending aorta, and aortic arch). Compared with those in AngII+Vehicle group, the mice in the CCB groups exhibited significant aortic expansion after 14 days of treatment, which persisted for 21 days. The AngII+verapamil group presented the largest thoracic aortic diameter among the CCB groups (AngII+verapamil vs. AngII+vehicle; Aortic root, 2.72 ± 0.10 vs. 2.18 ± 0.04 mm, P=0.006; Ascending aorta, 2.25 ± 0.12 vs. 1.76 ± 0.02 mm, P=0.043; Aortic arch, 1.93 ± 0.05 vs. 1.64 ± 0.02 mm, P=0.006) (Fig. 2c–f). Additionally, considering the potential impact of VSMC contractility changes on vascular mechanical properties15,19,29, we measured the thoracic root-to-descending pulse wave velocity (rd-PWV) and aortic strain in mice using M-mode ultrasound. These two indicators jointly reflect vascular stiffness and elasticity (Fig. 2g). Interestingly, compared with the AngII+Vehicle group, CCB treatment significantly increased the rd-PWV and decreased the ascending aortic strain after 7 days, with these differences becoming more pronounced after 14 and 21 days (Fig. 2h, i). These data indicate that CCB treatment increased aortic stiffness in AngII-infused mice, preceding aortic dilation. After 21 days of CCB treatment, the mice were sacrificed, and no significant differences in body weight or plasma lipid levels were observed among the groups (Supplementary Table 14). Ex vivo measurements revealed that both amlodipine and nifedipine significantly increased the maximum diameter of the descending aorta compared with that in AngII+Vehicle group. While the AngII+diltiazem and AngII+verapamil groups tended toward larger aortic root diameters, but this difference did not reach statistical significance compared with AngII+Vehicle group. All four CCBs significantly increased the diameters of the ascending aorta and aortic arch (Fig. 2j–m). Further Elastic Van Gieson (EVG) staining revealed that CCBs significantly exacerbated elastic fiber breakage and degradation in thoracic aortas (Supplementary Fig. 1b). Taken together, these results suggested that all types of CCBs exacerbated TAA (thoracic aortic aneurysm) expansion in AngII-infused mice. Meanwhile, ultrasound monitoring revealed a decrease in abdominal aortic strain as early as 7 days after CCB treatment, indicating increased vascular stiffness compared to the AngII+Vehicle group (Fig. 3a, b). After 14 days of CCB treatment, significant diameter expansion was observed in the suprarenal abdominal aorta, with the AngII+verapamil groups showing the largest diameter (Fig. 3c). Finally, CCB treatment not only increased the maximum abdominal aortic diameter and the incidence of AAA (AngII+Vehicle, 0% [0/13]; AngII+amlodipine, 41.7% [5/12]; AngII+nifedipine, 41.7% [5/12]; AngII+diltiazem, 45.5% [5/11]; AngII+verapamil, 63.6% [7/11]) (Fig. 3d–f), but also consistently exacerbated elastic fiber degradation in the abdominal aorta (Supplementary Fig. 1c). Moreover, in an elastase-induced infrarenal AAA model, compared with the Elastase+Vehicle group, CCB treatment further increased the maximum aortic diameter and exacerbated elastic fiber degradation with no significant differences in SBP or body weight among the groups (Fig. 4a–f and Supplementary Table 15). Previous study revealed that CCBs increased ERK1/2 signal activation to accelerate aorta expansion in Marfan Syndrome27. Here, we observed that in AngII-pretreated VSMCs, amlodipine and verapamil significantly increased the expression of inflammatory factors (IL-6 and MCP-1) while activating ERK1/2 signal. Furthermore, treatment with the ERK1/2 inhibitor U0126 significantly blocked the CCB-induced upregulation of inflammation (Supplementary Fig. 1d, e). Taken together, these data revealed that CCBs promote the progression of both TAA and AAA.

Losartan does not attenuate thoracic and abdominal aortic aneurysm formation in AngII-infused mouse models
Next, another class of antihypertensive drugs, angiotensin II type 1 receptor blockers (ARBs), on the development of AAD was evaluated in the AngII-perfused mouse model. To maintain an appropriate observation window, 24-week-old male C57BL/6J mice were used. First, these mice were infused with AngII (1000 ng/kg/min) to induce hypertension. Seven days later, mice that died from aneurysm rupture or were confirmed to have aneurysms by ultrasound were excluded from the study. The remaining hypertensive mice received intraperitoneal losartan (10 mg/kg/day) for 21 consecutive days (Supplementary Fig. 2a and Supplementary Table 16). As shown in Supplementary Fig. 2b, AngII-induced hypertension was significantly reduced within 7 days of losartan administration. At the end of the experiment, there were no significant differences between the two groups in terms of maximum diameter of ascending aorta (2.04 ± 0.10 vs. 2.02 ± 0.12 mm), aortic arch (1.76 ± 0.06 vs. 1.67 ± 0.07 mm) and descending aorta (1.70 ± 0.06 vs. 1.56 ± 0.06 mm) (Supplementary Fig. 2c–f). For abdominal aorta, no significant differences were observed between AngII+Vehicle and AngII+Losartan group in terms of the AAA incidence (AngII+Vehicle, 50% [5/10]; AngII+Losartan, 60% [6/10]) or maximum diameter of abdominal aorta (1.78 ± 0.18 vs. 1.73 ± 0.19 mm) (Supplementary Fig. 2g-i). These results suggest that losartan may not influence the incidence or progression of aortic aneurysm in hypertensive mice.

CCBs aggravated BAPN-induced TAAD in mice
Next, we applied a BAPN-induced mouse model of TAAD. Three-week-old C57BL/6J male mice were administered freshly prepared BAPN solution in the drinking water at a dosage of 0.5 g/kg per day for 28 days. After 7 days of BAPN treatment, the mice were injected daily with either vehicle or one of four CCBs. The control mice received normal drinking water (Fig. 5a). There were no observable differences in body weight and water intake among the BAPN groups (Supplementary Table 17). As a result, BAPN treatment led to rupture and premature death in 13.3% of the mice. Amlodipine, nifedipine, and verapamil increased the rupture and death rates, whereas diltiazem did not significantly affect mortality (Fig. 5b). Compared with the BAPN+Vehicle group, all four CCBs significantly increased the incidence of TAAD (BAPN+Vehicle, 46.7% [7/15]; BAPN+amlodipine, 100% [10/10]; BAPN+nifedipine, 100% [7/7]; BAPN+diltiazem, 85.7% [6/7]; BAPN+verapamil, 90% [9/10]) (Fig. 5c, d). Ex vivo measurements revealed significant expansion of the ascending aorta, aortic arch, and descending aorta in all CCB-treated groups compared with that in BAPN+Vehicle group (Supplementary Fig. 3a–c). EVG staining further revealed that CCBs markedly exacerbated elastic fiber breakage and degradation (Fig. 5e and Supplementary Fig. 3d). In a parallel experiment, early changes in thoracic aortic mechanical properties were detected using ultrasound (Supplementary Fig. 3e), which revealed a significant increase in rd-PWV and decrease in ascending aortic strain after as early as 3 days after the start of amlodipine and verapamil treatment. These effects occurred before any observable aortic dilation and intensified after 7 days (Fig. 5f, g and Supplementary Fig. 3f–j). Thus, CCBs aggravated BAPN-induced TAAD and aortic stiffness in mice.

CCBs application limited aortic regression in patients with Type B aortic dissection patients following endovascular therapy
Enabled by the GUEST study (NCT04765605), a single-arm, prospective, multicenter clinical trial that primarily evaluated the treatment efficacy of a novel stent graft system for type B aortic dissection (TBAD) (Fig. 6a), we further conducted a secondary analysis to assess the impact of CCB use on aortic expansion. In accordance with the study design, participants underwent computed tomography angiography (CTA) at 1 month, 6 months, and annually after thoracic endovascular aortic repair (TEVAR). Among the 120 enrolled participants, 95 were included in the final analysis, with 69 using CCBs, either exclusively or in combination with other antihypertensive medications and 26 using other antihypertensive medication (Fig. 6b and Supplementary Table 18). Baseline characteristics, preoperative laboratory results, and initial aortic morphology are shown in Supplementary Tables 19, 20. The alterations in aortic diameters, including those in Ishimaru zone 3 and zone 4, and the maximum descending thoracic aortic diameter (DTA max), as well as volumes in zones 3-4, zone 5, and zones 3-5, were measured and analyzed in strict accordance with the established reporting standards30 (Fig. 6c and Supplementary Fig. 4a).

As a result, at the 1-year follow-up, the growth rates (%) for all indicators—including zone 3, zone 4, and DTA max diameters, as well as zones 3-4, zone 5, and zones 3-5 volumes—were significantly higher in CCB users compared to those on other antihypertensive medication. By the 2-year follow-up, these elevated growth rates remained significant for all indicators except for the zone 3 diameter (Fig. 6d). Using mixed-effects models, we observed that CCB users had greater increases in DTA max zone 4, and zone 3 diameters, as well as larger zones 3-5, zone 5, and zones 3-4 volumes, compared with those in patients using other antihypertensive medications (P<0.05 for all, Supplementary Fig. 4b–g). After further adjustment for variables such as the aortic dissection phase, false lumen extension, thrombus status in the false lumen, intimal tear number and size, and initial aortic diameter and volume, differences in diameter between the two groups remained significant (zone 4: P=0.028, DTA max: P=0.002) and became more pronounced over time. Similarly, the adjusted differences in zones 3-4 (P=0.036), zone 5 (P=0.006), and zones 3-5 (P=0.005) volumes were also significant and increased throughout the 2-year period (Fig. 6e, f). These findings suggest that TBAD patients who undergo TEVAR and subsequently use CCBs may experience worsened aortic regression/shrinkage.

PRKG1 silencing alleviates CCB-induced exacerbation of AAD in mice
VSMC contractility is driven primarily by the phosphorylation of the myosin regulatory light chain (p-MLC), which is positively regulated by the intracellular Ca2+/calmodulin-myosin light chain kinase (MLCK) pathway. In contrast, cGMP-dependent protein kinase type I (PRKG1) enhances the activity of myosin light chain phosphatase (MLCP) by inhibiting myosin phosphatase target subunit 1 (MYPT1) phosphorylation, thereby counteracting the phosphorylation of MLC (Fig. 7a)18,31. Notably, a gain-of-function mutation in PRKG1 has been associated with spontaneous AAD in both patients and transgenic mice32,33. We did observe a significant decrease in p-MLC and p-MYPT1 levels in the aortas of verapamil-treated mice, in both AngII-induced and BAPN-induced AAD models (Fig. 7b, c). Therefore, we hypothesized that genetically inhibiting the Prkg1 to restore VSMC contractility may mitigate CCB-induced aggravation of AAD.

An adeno-associated virus 9 (AAV9) vector containing SM22α promoter was designed to specifically knockdown Prkg1 in VSMCs (Fig. 7d). Sixteen-week-old C57BL/6J mice were intravenously injected with either AAV9-Sh Scramble or AAV9-Sh Prkg1. After 7 days, the mice received subcutaneous infusions of AngII for 28 days, followed by daily intraperitoneal injections of either vehicle or verapamil starting 7 days after AngII treatment (Fig. 7e). Ultrasound results revealed that verapamil significantly increased aortic stiffness in AAV9-Sh Scramble-treated mice, but the differences were not observed in the AAV9-Sh Prkg1-treated mice (Fig. 7f–h). As expected, in AAV9-Sh Scramble-treated mice, verapamil significantly increased the maximum diameter of the thoracic aorta (including the aortic root, ascending aorta, and aortic arch) (Fig. 7i–l), the incidence of AAA (Fig. 7m, n), the maximum diameter of the abdominal aorta (Fig. 7o) and elastic fiber degradation (Supplementary Fig. 5a–d) compared with those associated with vehicle treatment. However, these differences were no longer present in the AAV9-Sh Prkg1-treated mice. Similarly, in AAV9-Sh Scramble or AAV9-Sh Prkg1-treated mice subjected to BAPN treatment, verapamil significantly increased the incidence of TAAD in the AAV9-Sh Scramble group (Sh Scramble+Vehicle: 45% [9/20]; Sh Scramble+Verapamil: 81.25% [13/16]), whereas Prkg1 knockdown abolished these effects (Sh Prkg1+Vehicle: 50% [5/10]; Sh Prkg1+Verapamil: 40% [4/10]) (Supplementary Fig. 6a–c). Additionally, ex vivo measurements (Supplementary Fig. 6d–f) and EVG staining (Supplementary Fig. 6g, h) revealed that Prkg1 knockdown mitigated CCB-induced aortic expansion and elastic fiber degradation, respectively. Detailed characteristics of these mice are summarized in Supplementary Tables 21, 22. Taken together, these data indicate that inhibiting the PRKG1-MLCP pathway to restore VSMC contractility can mitigate CCB-aggravated AAD formation.

Methods
UKB study population
The UK Biobank (UKB) is a large population-based cohort, including over 500,000 volunteers aged between 40 and 69 years across England, Scotland, and Wales recruited between 2006 and 2010 (Details could be found at https://www.ukbiobank.ac.uk). All UKB participants provided informed consent via an electronic signature at the beginning of the process and the UKB had ethical approval from the North West Multi-Center Research Ethics Committee (REC ref. 16:/NW/0274). Included were 501,878 participants in this study after removing participants who withdrew from the UKB and prevalent aortic aneurysm and dissection (AAD). We also investigated the association between subtypes of CCBs and AAD in the participants with prevalent hypertension (n=142,419) and the association between the CCBs use and all-cause mortality in the participants with prevalent aortic aneurysm and dissection (n=509).

Assessment of exposure
Baseline hypertension was defined through self-reported hypertension during nurse interview, and self-reported questionnaires regarding high blood pressure diagnosed by doctor, hospital data via international classification of diseases (ICD) codes for essential (primary) hypertension, hypertensive disease with or without heart failure, hypertensive heart and renal diseases, and secondary hypertension according to previous study76 (Supplementary Table 1). A high concordance was observed between self-reported (either self-reported hypertension during nurse interview or self-reported high blood pressure diagnosed by doctor) and hypertension identified through hospital records, with more than 94% of individuals who self-reported hypertension also having a physician-confirmed diagnosis based on ICD codes. We also adopted a broader definition of hypertension by further incorporating antihypertensive medications use in the sensitivity analysis. Antihypertensive medications regarding calcium channel blocker use (Supplementary Table 2) and other blood pressure lowering medications including renin-angiotensin system inhibitors, beta blockers, diuretic, or multiple antihypertensive classes were assessed at baseline76,77 (Supplementary Table 3). All self-reported medications were recorded and subsequently confirmed by a trained nurse in an interview conducted during the same visit. Additionally, participants who reported taking a blood pressure medication (via Field IDs 6153 and 6177) without using CCBs were categorized under the “other antihypertensive medications” group (excluding CCB use). Any one reporting concurrent CCB use was categorized into the CCBs group, including those using CCBs exclusively or in combination with other antihypertensive medications. There was no recorded information concerning the dosage, frequency, or duration of use for medication. We categorized groups according to hypertension status and antihypertension medications (Fig. 1).

Assessment of covariates
At baseline, data on sociodemographic factors (age, sex, education, and ethnicity), BMI, and lifestyle factors (smoking status and alcohol use) were collected through questionnaires and verbal interviews. Total cholesterol, total triglycerides, and systolic blood pressure were also analyzed in this study, regarding to the risk of AAD. Specifically, serum lipid levels were measured by biochemical assays from the blood samples collected at baseline by Beckman Coulter AU5800 Platform. Total cholesterol and total triglycerides were analyzed using glycerol-3-phosphate (GPO)-peroxidase (POD) chromogenic method. The resting blood pressure was measured by Omron 705 IT electronic blood pressure monitor. During the blood pressure measurement, the participant was instructed to sit with their feet parallel, toes pointing forward, and the soles of both feet flat on the floor. The right arm was only used if the left was not feasible. The mean value of blood pressure readings would be calculated and used in this analysis if there were more than one measurement within a few minutes available; otherwise, electronic blood pressure monitor values were preferred, and manual readings would be used if there were no electronic blood pressure readings.

Assessment of outcome
AAD cases and its subtypes including thoracic aortic aneurysm and dissection (TAAD) and abdominal aortic aneurysm (AAA) were identified from the UKB using hospital inpatient records where these conditions were documented as either primary or secondary diagnosis, or linkage to death register data using the International Classification of Disease (ICD) coding 10 (Supplementary Table 4).

Materials
Amlodipine (HY-B0317), Nifedipine (HY-B0284), Diltiazem (HY-14656), Verapamil (HY-14275), Losartan (HY-17512), human angiotensin II (HY13948) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Osmotic pumps (2004W) were purchased from RWD Life Science Co (Shenzhen, Guangdong, China). Elastase, porcine pancreas (HY-P2974) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). β-aminopropionitrile monofumarate (BAPN, A3134) was purchased from Sigma Aldrich (St. Louis, MO, USA). PEG200 (P8490), Tween 80 (T8360) and protein phosphatase inhibitor (P1260) were purchased from Solarbio (Beijing, China). Antibodies against GAPDH (10494), PRKG1 (21646) were purchased from ProteinTech (Chicago, IL, USA). Antibody against p-MLC (3671), MLC (8505), p-MYPT1 (4563), p-ERK1/2 (9101) was purchased from Cell signaling Technology (Boston, MA, USA). ERK1/2 (T40071F) was purchased from Almart (Shanghai, China).

Animals
Animal studies were conducted in compliance with the guidelines of the Institutional Animal Care and Use Committee of Peking University Health Science Center and were approved by the Biomedical Ethics Committee of Peking University. SPF mice of C57BL/6J were purchased from the Experimental Animal Science Center and raised in the Experimental Animal Science Center of Peking University Health Science Center, Beijing, China. The mice were randomly assigned to different groups and housed in cages with 4–6 mice per cage with unrestricted access to rodent feed and water. Specifically, a light-dark cycle was established with lights on from 8 a.m. to 8 p.m., while maintaining a temperature range of 21-24°C and humidity levels between 40–70%. Mice for surgeries were anesthetized with isoflurane (1.5%–2%). All animals were anesthetized with 1.25% avertin before being sacrificed. For plasma samples, blood was collected from the heart with heparin sodium (250 U/ml), followed by centrifugation for 10 min at 3000 rpm. For aorta protein samples, mice were perfused with ice-cold saline, and then the aorta was isolated. For aortic morphology studies, the mice were successively perfused with 10% KCl, 10× PBS, and 4% paraformaldehyde. Then, the aortas were dissected from the root to the iliac bifurcation in all animal models for further analysis.

Drugs dissolution and injection
The doses of drugs used in the experiment were converted from clinical recommended human doses to mouse doses according to the FDA’s guidance for dose translation (https://www.fda.gov/media/72309/download, conversion factor=12.3). The specific doses were as follows: amlodipine (1 mg/kg/day), nifedipine (20 mg/kg/day), diltiazem (8 mg/kg/day) and verapamil (12 mg/kg/day), losartan (10 mg/kg/day). The dissolution protocol used a gradient of DMSO, PEG 200, Tween 80, and saline. The final CCB injection solution consisted of 2% DMSO, 8% PEG 200, 1% Tween 80, and 89% saline. Experimental mice were administered the specified dose of CCBs or vehicle daily via intraperitoneal injection based on body weight, continuing until the sacrifice endpoint.

Angiotensin II-induced aortic aneurysm mouse model
Sixteen-week-old C57BL/6J mice were subcutaneously infused with minipumps containing AngII (1000 ng/kg·min−1) for 28 days. After 7 days of AngII infusion, images of mice thoracic and abdominal aorta were obtained via ultrasound instrument. During hypertension establishment phase, (1) mice died due to aortic aneurysm rupture; or (2) mice diagnosed with developed aorta aneurysm by ultrasound imaging (TAA or AAA: defined as a segmental aortic diameter >1.5 times that of the adjacent normal aorta) were excluded from the study and those hypertensive mice without AAD were remained. Subsequently, those hypertensive mice without AAD were daily injected with vehicle or one of four CCBs: amlodipine, nifedipine, diltiazem or verapamil (Supplementary Fig. 1a). SBP was recorded at 0, 3, 7, 14, 21 days after vehicle/CCBs treatment. Root-descending aorta pulse wave velocity, ascending and abdominal aorta strain, thoracic and abdominal aorta diameters were detected at 0, 7, 14, 21 days after vehicle/CCBs treatment by ultrasound. For losartan experiment, six month-old C57BL/6J mice were infused with AngII (1000 ng/kg·min−1) for 28 days. After 7 days of infusion, the mice were daily injected with vehicle or losartan (10 mg/kg/day). SBP was recorded at 0, 7, 14, 21 days after vehicle/losartan treatment.

At the sacrificed endpoint, aortas were dissected for ex vivo measurements of the maximal thoracic aorta diameter (ascending aorta, aortic arch, descending aorta) and abdominal aorta diameter. Aortic aneurysm was evaluated in the suprarenal abdominal aorta (referred to as AAA) and the aortic root and ascending aorta (referred to as TAA). The maximal diameters of the root, ascending and suprarenal aortas were calculated by statistical analysis, and the aortic aneurysm was defined as an aorta with a diameter more than 1.5 times larger than its adjacent normal aorta.

Elastase-induced abdominal aorta aneurysm mouse model
Eight-week-old C57BL/6 mice were used for surgery. Briefly, the mice were anesthetized and the infrarenal aortas were exposed, isolated, and wrapped with sterile cotton, which was soaked with 25 μl of elastase (10 mg/ml) for 40 min. PBS was used as negative control. Then, the elastase-soaked cotton was removed, and 0.9% NaCl was used to perfuse the abdominal cavity before suturing. Subsequently, these mice were administered intraperitoneal injections of vehicle or four CCBs for 14 days.

At sacrificed endpoint, systolic blood pressures were measured and aortas were dissected for ex vivo measurements of the maximal abdominal aorta diameter.

BAPN-induced TAAD mouse model
Three-week-old C57BL/6 mice were treated with 0.3% BAPN in drinking water (0.5 g/kg per day) for 28 days. The drinking water was refreshed every 3 days. Normal drinking was designed as negative control. After 1 week, the mice were daily injected with vehicle or four CCBs. It is important to note that due to the small size of the TAAD model mice and the potential risk of TAAD rupture induced by stress during blood pressure measurement, this model is not suitable for recording SBP.

At the sacrificed endpoint, aortas were dissected for ex vivo measurements of the maximal thoracic aorta diameter (ascending aorta, aortic arch and descending aorta). The incidence rate of TAAD and related mortality were recorded. TAAD was defined as thoracic aortic dissection (aortic false lumen formation) and/or thoracic aortic aneurysm (at least 1.5 times extension of aortic diameter). Overall, any of the following criteria was defined as TAAD: 1. The deceased mice displayed blood clotting in the thoracic cavity. 2. EVG staining revealed aortic false lumen formation. 3. >1.5 times extension of aortic diameter compare to negative control.

In another parallel BAPN experiment, ultrasound was used to measure root-descending aorta pulse wave velocity, ascending aorta strain, thoracic aorta diameters at 0, 3, 7 days after vehicle/amlodipine/verapamil treatment.

Mouse blood pressure measurement
Mouse blood pressure was measured according to the previous study78. Briefly, a noninvasive computerized CODA tail-cuff blood pressure system (Kent Scientific, Torrington, CT, USA) was used to measure mouse SBP. The equipment was maintained in a clean state, free from foreign scents or blood odors. The investigator was blinded to the experimental groups when performing the measurements, and the mice were tested in a randomized order. All mice underwent training sessions from 1 to 4 PM on 7 consecutive days to become accustomed to the tail-cuff procedure.

Mouse aorta ultrasound
Vevo LAZR system (FUJIFILM VisualSonics, Canada) was used to measure aorta parameters in mice, including thoracic aortic diameter, abdominal aortic diameter, root to descending pulse wave velocity (rd-PWV), ascending aorta and abdominal aorta circumferential strain. Mice were anaesthetized by 1.5–2% isoflurane and placed on the platform that carries electrocardiogram (ECG) electrodes. Conductive gel was applied to the electrodes on the animal platform, and the mouse’s paws were secured onto the electrode pads with tape to ensure accurate ECG acquisition. The mouse’s body temperature and respiratory rate were maintained, with the heart rate kept within the range of 400–550 bpm during the acquisition. MX transducer MX550D (centre transmit: 40 MHz; axial resolution: 40 μm) was used. The thoracic and abdominal aorta diameters were recorded by B-mode ultrasonography. For aorta diameter, images of the aortic arch cross-section (the operation table was tilted 15° to the right, and the probe arm was tilted 30-45° to the left) and abdominal aorta sagittal section (the probe was positioned perpendicular to the animal) were obtained. Rd-PWV was measured using ultrasound imaging by B-mode and Doppler-Mode. The vascular pulse-wave Doppler tracing was recorded at the position of root (position 1, d0). The time from the R point of QRS to the onset of the Doppler waveform was measured (T1). Then another position in the descending aorta was selected and the pulse-wave Doppler tracing was recoded (position 2, d). The time from the R point of QRS to the onset of the Doppler waveform was measured (T2). The distance between position 1 and position 2 was measured (distance). Rd-PWV is calculated as: distance/(T2–T1) (m/s). The values for T1 and T2 were the average of three to five measurements. For ascending aorta and abdominal aorta circumferential strain, the systolic diameter (Ds) and diastolic diameter (Dd) were measured, and circumferential cyclic strain was calculated as (Ds-Dd)/Dd. The values for Ds and Dd were the average of three to five measurements.

Blood biochemical analysis
Blood samples were collected and underwent centrifugation. The quantification of plasma cholesterol and triglyceride levels was performed using commercially available kits (#100000220, ZhongSheng Biotechnology).

Elastin Van Gieson staining
Cryosections of the lesion aorta (8 μm thick) were analyzed by elastic Van Gieson staining (BASO Precision Optics Ltd., Taiwan, China) according to the manufacturer’s protocol for elastin assessment. Elastin degradation graded as grade 1, <25% degradation; grade 2, 26% to 50% degradation; grade 3, 51% to 75% degradation; grade 4, >75% degradation.

Adeno-associated virus 9 (AAV9) production and infection
A plasmid (GV706) encoding promoter SM22α, EGFP and shRNA targeting mouse Prkg1 was engineered by cloning the following shRNA into the AAV9 vector:
Sh Prkg1 (sense) 5’-GATCCCCGGAGAATCTCATCCTAGATCTCGAGATCTAGGATGAGATTCTCCGGTTTTTGC-3’;
Sh Prkg1 (antisense) 5’-GGCCGCAAAAACCGGAGAATCTCATCCTAGATCTCGGATCTAGGATGAGATTCTCCGGG-3’;
The sequence of the mouse Prkg1 shRNA was referred to the previous study55. Packaging and producing work were performed by GENE company (Shanghai, China). For in vivo infection experiments, every mouse was intravenously administered AAV9-Sh Scramble and AAV9-Sh Prkg1 with 2×1011 v.g. titer. Infection efficiency was analyzed in thoracic aorta samples by western blot.

Western Blot
Mouse tissues or cells were lysed in RIPA buffer (P0013B, Beyotime, Beijing, China) supplemented with protein phosphatase inhibitors (1:100). Protein concentrations were quantified using a BCA Protein Assay Kit (Thermo Scientific). Equal amounts of total proteins were resolved on 8% or 12% SDS-PAGE gels transferred onto nitrocellulose filter membranes (Pall, Port Washington, NY, USA). The membranes were blocked with 5% BSA and then incubated with the primary antibody and the IRDye 700/800DX-conjugated secondary antibody (Rockland Inc., Gilbertsville, PA, USA). The immunofluorescent signal was obtained using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).

Cell Culture
Primary rat VSMCs were isolated from the thoracic aortas of 150 to 180 g male Sprague-Dawley rats by collagenase digestion. VSMCs were cultured in low glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Primary VSMCs at passages 3 to 6 were used for all experiments. VSMCs were maintained at 37°C in a humidified atmosphere with 5% CO2.

TBAD study and Anatomical characteristics
TBAD clinical data is from the prospective multicenter clinical trial, conducted in accordance with the ethical standards of the Institutional Review Board of the Chinese PLA General Hospital (2020010), was carried out in 18 tertiary hospitals across China. The study was registered with ClinicalTrials.gov under the identifier NCT04765605.

For patient inclusion and exclusion criteria, we excluded deceased patients for whom CTA data could no longer be obtained and patients with major postoperative complications, such as stroke, respiratory failure, retrograde type A aortic dissection, re-intervention, stent-induced new entry, stent migration, stenosis or occlusion of the LSA branch stent, and spinal cord ischemia, due to their poor health status in subsequent follow-ups. In addition, several patients who were not hypertensive were also excluded from this study.

For antihypertensive medication, we confirmed and recorded the patients’ use of antihypertensive medication through discharge medication records, subsequent follow-up information from outpatient visits at the central hospital, and telephone follow-ups.

For CTA measurements, briefly, a series of morphological parameters of TBAD were measured on the preoperative and postoperative CTA images. The measurement protocol strictly followed the reporting standards for TBAD recommended by the Society for Vascular Surgery and Society of Thoracic Surgeons30. All measurements were obtained using post-processing images software (3Mensio Workstation version 10.4, Maastricht, the Netherlands) that enabled aortic centre lumen line extraction and reconstruction. Aortic diameter was measured in three fixed planes, including: (1) 2 cm distal to the left subclavian artery (LSA); (2) the middle point of the descending thoracic aorta (DTA) and (3) the superior border of the celiac artery (CA). Additionally, the maximal diameter of the DTA was also recorded. Suggested by the Society for Vascular Surgery and Society of Thoracic Surgeons, the aortic diameter was measured from the outer aortic wall to outer aortic wall, and the diameters of the true lumen (TL) and false lumen (FL) on these four interested observed planes were simultaneously measured. The volumes of the DTA (Ishimaru zones 3-4 and zone 5) was also measured by using the “volume segmentation function” of the post-processing software 3Mensio Vascular. The descending thoracic aorta is anatomically divided into the Ishimaru zones 3-4 and zone 5 (Supplementary Fig. 4a).

Statistical analysis
For UKB data, we summarized the baseline characteristics of the study stratified by hypertension and CCBs use or other antihypertensive medication, or hypertension without antihypertensive medication use, or no hypertension (Fig. 1 and Supplementary Table 5). Person-time was calculated from the baseline to the occurrence of study outcomes, death, or the end of follow-up (October 31, 2022 [England], July 31, 2021 [Scotland], and February 28, 2018 [Wales]), whichever came first. Cox proportional hazards regression models were applied to examine the risk of AAD or AAD subtypes, adjusting for age, sex, BMI, total cholesterol, triglycerides, smoking status, education, ethnicity, and systolic blood pressure. Sensitivity analyses were conducted to minimize the bias: (1) a broader definition of hypertension was applied; (2) restricted to participants without coronary artery disease to address the issue that coronary artery disease might confound the association between CCBs use and AAD; (3) the Fine & Gray competing risk model to account for the presence of competing events (death from all causes except for AAD). We tested the proportional hazard assumption with Schoenfeld residuals and did not detect any significant violation of this assumption. Among participants with hypertension, mixed-effects linear models with unstructured variance structure were used to analyze the effect of groups (without any antihypertensive medications, CCBs use, and other antihypertensive medications excluding CCBs use) on systolic/diastolic blood pressure, with participants treated as random effects, group, time (baseline or follow-up), and their interaction as fixed effects, adjusting for age, sex, BMI, total cholesterol, triglycerides, smoking status, education, ethnicity, and baseline systolic/diastolic blood pressure. Estimated marginal means and 95% CI were the adjusted values from the models and post hoc pairwise comparisons used Bonferroni correction. Statistical analyses for the UKB were performed using R version 3.6.3.

For experiments data, statistical analyses were performed using GraphPad Prism 8.0 software (San Diego, CA, USA). Data are expressed as mean ± SEM. The details of the statistical analysis used in each experiment are presented in the corresponding figure legends. For statistical comparisons, we first evaluated whether the data were normally distributed using the Shapiro Wilk normality test. For two groups, we used unpaired Student’s t test for normally distributed data. If the data was skewed distribution, we used Mann-Whitney test. For more than two groups with one variable, we used one-way ANOVA followed by post hoc analysis for comparisons among normally distributed data. Specifically, Dunnett’s multiple comparisons test was used to compare each group with a control group, while Tukey’s multiple comparisons test was used to compare each group with every other group. If the data equal variances were not assumed, Brown-Forsythe and Welch ANOVA test was used with Dunnett T3 multiple comparisons test (compare each group with a control group) or Games-Howell multiple comparisons test (compare each group with every other group). For more than two groups with two variables, we used two-way ANOVA (mixed model) among normally distributed data. Specifically, Dunnett’s multiple comparisons test was used to compare one variable within each another variable to control group. If the data equal variances were not assumed, Geisser-Greenhouse correction was used. Nonparametric tests (Kruskal-Walli’s test) were used when the data were not normally distributed. In addition, Fisher’s test was used for contingency data and Log-rank test was used Kaplan-Meier curve.

For human study, statistical analyses were performed using GraphPad Prism 8.0 software (San Diego, CA, USA) and SPSS Statistics 26 (IBM, NY, USA). Data are expressed as mean ± SEM or percentages. For statistical comparisons, unpaired Student’s t test and χ test (Fisher’s test) was used for the comparison of patient’s baseline characteristics, CTA measurements and diameters or volumes growth rates at 1- or 2-year post-TEVAR. Mixed-effects model without adjustment was used to compare whether there are statistically significant differences in the trends of diameters and volumes between the two groups over a 2-year follow-up period. This model accounts for repeated measures within subjects and evaluates changes over time without adjusting for potential confounding variables. Furthermore, we applied a general linear mixed-effects model, adjusting for potential influencing factors, including age (years), sex (men; women), systolic blood pressure (mmHg), family history of cardiovascular disease (yes; no), diabetes (yes; no), hyperlipidemia (yes; no), coronary heart disease (yes; no), false lumen length grades (1-8), total false lumen thrombus score (0; 1; 2; 3 levels for each zone and combined score for all zones), number of distal tears (zone 5-9), first tear diameter (mm), and interactions of these factors with CCB use. We then compared whether the trends of diameters and volumes between the two groups over the 2-year follow-up period showed statistically significant differences. Additionally, we assessed whether the differences in diameter and volume between the two groups at preoperative and postoperative time points (1, 6, 12, and 24 months) were statistically significant.

In all analyses, P-value<0.05 was regarded as statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Ma et al. evaluated the role of calcium channel blockers (CCBs) in aortic aneurysm and dissection (AAD).\nThe authors utilized a large-scale population-based cohort and found that various subtypes of CCBs were associated with\nan increased risk of incident AAD. Furthermore, the findings were supported by multiple animal models, including the Ang II-\ninduced aortic aneurysm model, the elastase-induced abdominal aortic aneurysm model, and the BAPN-induced thoracic\naneurysm and dissection model, all of which suggested that CCBs may promote AAD incidence and increase aortic\nstiffness. The authors also conducted a secondary analysis of a multicenter clinical trial, revealing that postoperative use of\nCCBs in type B aortic dissection (TBAD) patients was associated with impaired aortic shrinkage.While prior studies have\nlinked VSMC contraction inhibition to AAD, direct population-based evidence remains lacking. Using the UKB prospective\ncohort and a well-designed animal model, this work clearly shows the essential role of maintaining VSMC contractility in\naortic homeostasis. Findings from this study are highly significant because many hypertensive patients are using CCBs to\nreduce blood pressure. Therefore, these findings will offer new insights for guiding clinical medication in patients with\nhypertension. Overall, the manuscript integrates population analysis with rodent model validation to address key vascular\nbiology questions, offering significant innovation. Below are several issues needed to be raised.\nMajor Comments\n1. In the UK Biobank data analysis, the \"other antihypertensive medication\" group showed no significant association with\nincreased or decreased risks of incident AAD, TAAD, or AAA. This finding requires discussion. Additionally, incorporating\nrelevant control experiments in animal models—such as testing the effects of other anti-hypertension drugs, for example,\nARBs or ACEIs, in rodent AAD models—may provide deeper insights beyond the current observational results.\n2. In the Ang II-induced aortic aneurysm model, the authors first induced hypertension in mice and then excluded animals\nthat had already developed aneurysms during the hypertensive phase using ultrasound, followed by initiating CCB\nintervention. This experimental design mirrors the clinical sequence of antihypertensive treatment in hypertensive patients.\nHowever, it is noted that after exclusion, no aneurysms were observed in the Ang II + vehicle group, this may lead to\nmisunderstanding. The authors should clarify the exclusion criteria and the number of animals excluded in the manuscript.\n3.The authors conducted a secondary analysis of a multicenter clinical study and found that CCB use was associated with\nworsened aortic regression/shrinkage in postoperative TBAD patients. However, how should \"worsened aortic\nregression/shrinkage\" be interpreted? Does this phenomenon stem from reduced vascular tone caused by CCBs, or does it\nreflect more adverse pathological remodeling of the aorta? The authors did not provide sufficient mechanistic explanations\nor clinical implications. It is recommended that the authors elaborate on this point in the Discussion section, particularly in\nthe context of postoperative aortic remodeling.\n4.In animal AAD models, CCBs significantly increased aortic stiffness (elevated PWV and reduced aortic strain), which\nappeared to precede aortic dilation.Is this effect a direct action of CCBs or an early change accelerating disease\nprogression? Would administering CCBs to healthy mice produce similar effects? Additionally, the authors should discuss\nthe potential mechanisms underlying CCB-induced early changes in vascular stiffness.\n5. Please provide sex and gender for human and mice studies in the Method or Result section. Does the sex affect CCBs-\ninduced AAD?\nMinor Comments\n1. 1. In lines 63–64, the authors state that “there are no currently proven drugs to slow aortic expansion or prevent aortic\nrupture in AAD patients (except for Marfan Syndrome).” While β-blockers or ARBs are recommended for patients with Marfan\nsyndrome, not all studies consistently support the efficacy of these therapies. Therefore, this statement is not entirely\naccurate and should be revised to reflect the variability in current evidence.\n2. How was the dosage of CCBs determined in animal models? The manuscript would benefit from a more detailed\ndescription of the dosing strategy in the Methods section.\n3. In Figure 5, the representative EVG staining of the BAPN+Vehicle group does not appear to show substantial elastic fiber\nfragmentation or degradation. To enhance the reliability of histological interpretation, it is recommended that the authors\nprovide additional EVG-stained images for this group.\n4. In Extended Data Figure 6, the summary of antihypertensive use in the post-surgery phase could be expanded. Please\nprovide more details and discuss postoperative hypertensive medication in the Discussion section.", "reviewer_2": "The study under review presents a groundbreaking investigation into the potential association between calcium channel\nblockers (CCBs) and aortic aneurysm and dissection (AAD), two life-threatening cardiovascular conditions. The findings are\nof paramount importance, given the prevalence of hypertension and the widespread use of CCBs as a first-line treatment.\nThe study's large sample size and long follow-up period lend significant weight to its conclusions, which suggest that CCBs\nmay be linked to an increased risk of AAD. This revelation could have far-reaching implications for clinical practice,\npotentially altering the way clinicians approach the management of hypertension in patients at risk of AAD. However, there\nare several aspects that could be further refined to strengthen the study's conclusions.\nFirstly, the research on downstream mechanisms is relatively superficial. The study suggests that CCBs may affect AAD\nthrough their impact on vascular smooth muscle cells (VSMCs). However, more rigorous cell experiments are needed to\nconfirm this. It is unclear whether the effect is direct or if there are intermediate factors involved. For instance, CCBs might\ninfluence intracellular calcium signaling pathways in VSMCs, which in turn affect cell contraction and proliferation. Detailed\nmolecular studies on VSMCs treated with CCBs could provide valuable insights into the underlying mechanisms.\nSecondly, it would be beneficial to investigate whether there is any known relationship between the calcium channels in\nVSMCs and PRKG1. PRKG1 has been shown to restore VSMC contractility and alleviate CCB-induced aortic stiffness and\nAAD incidence. Understanding the potential crosstalk between these two pathways could shed light on the complex\ninteractions within the vascular wall and help identify new therapeutic targets.\nThirdly, there are some studies that have shown CCBs may alleviate aortic aneurysm and dissection (J Mol Cell Cardiol. 2015 Aug 4;87:152–159; Front Pharmacol. 2023 Nov 10;14:1291900). The article should discuss these conflicting\nconclusions and explore the possible reasons. Differences in study populations, methodologies, and definitions of AAD\ncould contribute to the divergent results. A thorough comparison and analysis of these studies would provide a more\ncomprehensive understanding of the role of CCBs in AAD and help resolve the inconsistencies in the literature.\nIn conclusion, the study provides valuable insights into the potential relationship between CCBs and AAD, highlighting the\nneed for caution when prescribing CCBs to hypertensive patients at risk of AAD. The findings are particularly important\ngiven the widespread use of CCBs in clinical practice. Addressing the aforementioned points through a revision process\nwould significantly enhance the robustness and credibility of the findings."}, "author_response": "We would like to sincerely thank the Reviewers for their time, effort, and insightful feedback on our manuscript. We greatly\nappreciate the constructive comments and valuable suggestions, which have significantly helped us improve the clarity and scientific\nrigor of our study. We have carefully considered each comment and have revised the manuscript accordingly. Below, we provide a\ndetailed, point-by-point response to all the concerns raised. We hope that the revised version addresses all the issues and meets the\nstandards for publication.\nReviewer #1:\nIn this manuscript, Ma et al. evaluated the role of calcium channel blockers (CCBs) in aortic aneurysm and dissection (AAD). The\nauthors utilized a large-scale population-based cohort and found that various subtypes of CCBs were associated with an increased risk\nof incident AAD. Furthermore, the findings were supported by multiple animal models, including the Ang II-induced aortic aneurysm\nmodel, the elastase-induced abdominal aortic aneurysm model, and the BAPN-induced thoracic aneurysm and dissection model, all of\nwhich suggested that CCBs may promote AAD incidence and increase aortic stiffness. The authors also conducted a secondary analysis\nof a multicenter clinical trial, revealing that postoperative use of CCBs in type B aortic dissection (TBAD) patients was associated with\nimpaired aortic shrinkage.While prior studies have linked VSMC contraction inhibition to AAD, direct population-based evidence\nremains lacking. Using the UKB prospective cohort and a well-designed animal model, this work clearly shows the essential role of\nmaintaining VSMC contractility in aortic homeostasis. Findings from this study are highly significant because many hypertensive\npatients are using CCBs to reduce blood pressure. Therefore, these findings will offer new insights for guiding clinical medication in\npatients with hypertension. Overall, the manuscript integrates population analysis with rodent model validation to address key vascular\nbiology questions, offering significant innovation. Below are several issues needed to be raised.\nReply: we thank the reviewer for the positive feedback and constructive suggestions.\nMajor Comments\n1. In the UK Biobank data analysis, the “other antihypertensive medication” group showed no significant association with increased\nor decreased risks of incident AAD, TAAD, or AAA. This finding requires discussion. Additionally, incorporating relevant control\nexperiments in animal models—such as testing the effects of other anti-hypertension drugs, for example, ARBs or ACEIs, in rodent\nAAD models—may provide deeper insights beyond the current observational results.\nReply: We thank the reviewer for the constructive suggestions. At present, although several RCT studies have examined whether\nvarious antihypertensive drugs (such as ACEIs, ARBs, and β-blockers) can slow the progression of small abdominal aortic aneurysms\nor aortic aneurysms in patients with Marfan syndrome1-6, no studies have systematically explored how antihypertensive drug use affects\nthe new onset of AAD (including AAA, TAA, and TAD). The reason for this gap may be the relatively low incidence of AAD, which\nrequires a very large cohort size for relevant studies. In this study, we used a large-scale prospective general population cohort from\nUK Biobank and found that CCBs significantly increase the risk of new-onset AAD in hypertensive patients. At the same time, our\nresults showed that other classes of antihypertensive drugs (including ARBs, ACEIs, β-blockers, and diuretics, which were analyzed\ntogether in statistics) do not significantly affect the occurrence of AAD. This result is consistent with several previous RCT studies,\nwhich showed that ACEIs and ARBs do not reduce AAA growth rates1,2. However, we need to note that in this study, these “other\nantihypertensive medications” were grouped together as one of the controls for the CCB group. This may lead to mutual dilution of\ndifferent drug effects in statistics. As for how each class of antihypertensive drugs individually affects the new onset of AAD, further\nresearch is still needed.\nFollowing the reviewer’s suggestion, we have added a new experiment to test how the ARB drug losartan affects the incidence of\nAAD in AngII-induced hypertensive mice. To maintain an appropriate observation window, we used 24-week-old male C57BL/6J mice\n(16-week-old mice were used in the previous AngII+CCB model) to increase the AAD incidence in the control group. First, 24-week-\nold male C57BL/6J mice were infused with AngII (1000 ng/kg/min) to induce hypertension (Response Table 1). Seven days later, mice\nthat died from aneurysm rupture or had aneurysms confirmed by ultrasound were excluded from the experiment. The remaining\nhypertensive mice were intraperitoneally administered losartan (10 mg/kg/day) for 21 consecutive days. Blood pressure and aortic\nmorphology were monitored by a tail-cuff instrument and ultrasound, respectively. As shown in Response Figure 1, AngII-induced\nhypertension was significantly reduced within 7 days of losartan administration, indicating that losartan exerted its blood pressure-\nlowering effect. At the end of the experiment, 50% of mice in the AngII+Vehicle group developed AAA, while 60% of AAA in the\nAngII+losartan group. There were no significant differences between the two groups in terms of AAA incidence or the maximum\nvascular diameter of various aortic segments (ascending aorta, descending aorta, abdominal aorta). These results suggest that losartan\nmay not affect the development and progression of AAD in hypertensive individuals. We have added this data to Extended Data Figure\n2 in the revised version of the manuscript, please refer to page 9, line 194-210. This result is consistent with previous studies showing\nthat losartan does not alleviate AAD induced by elastase, BAPN, or DOCA7-9. However, in AngII models used in previous studies,\nlosartan or telmisartan was administered to animals simultaneously with AngII10-12. This directly led to the failure of hypertension\ninduction, so the observed alleviation of AAD development was predictable. Taken together, this newly added data serves as a control\nexperiment and strengthens our confirmation that CCBs, while lowering blood pressure, increase aortic pathology. As suggested by the\nreviewer, we have added a discussion of the effect of other antihypertensive medications on AAD in the manuscript: “Moreover, we\nobserved that other classes of antihypertensive drugs did not significantly affect AAD incidence—a result consistent with previous\nRCTs showing ACEIs and ARBs do not reduce AAA growth rates—though these ‘other antihypertensive medications’ may cause\nstatistical mutual dilution of different drug effects.” and “In a similar model, we also observed that losartan lowers blood pressure but\ndoes not affect the occurrence and progression of AAD. This finding is consistent with the results from the UKB analysis and previous\nstudies demonstrating that losartan does not attenuate AAD induced by elastase, BAPN, or DOCA.” please refer to Page 15, Line 337-\n341 and 348-351.\nResponse Figure 1. Losartan does not Attenuate Thoracic and Abdominal Aortic Aneurysm Formation in AngII-infused\nmouse model.\na, Experiment design. Six-month-old C57BL/6J mice were infused with AngII (1000 ng/kg·min-1) for 28 days. After 7 days of\ninfusion, the mice were daily injected with vehicle or losartan (10 mg/kg/day). Groups: Saline + vehicle (n=10); AngII + vehicle\n(n=10); AngII + losartan (n=10).\nb, The SBP of mice were recorded by tail-cuff blood pressure measurement at -7, 0, 7,14,21 days after vehicle/losartan treatment. *\nvs. AngII + Vehicle group.\nc, Representative ex vivo morphology of thoracic aortas from mice. Scale bar=2 mm.\nd-f, Maximal external diameters of ascending aorta, aortic arch and descending aorta.\ng, Representative ex vivo morphology of abdominal aortas from mice. Scale bar=2 mm.\nh-i, The incidence of AAA and maximal external diameters of abdominal aorta.\nb, d-f, i, Data are the mean ± SEM of each group. h, Data are presented as percentage (%). *P < 0.05; ns, no significant b, Two-way\nANOVA with Tukey multiple comparisons test. d, f, Kruskal-Wallis test with multiple comparisons; e, One-way ANOVA with\nHolm-Sidak multiple comparisons test; h, Fisher’s exact test. i, Brown-Forsythe and Welch ANOVA test with Dunnett T3 multiple\ncomparisons.\nAngII, angiotensin II; SBP, systolic blood pressure.\nResponse Table 1. Characteristics of mice (AngII-model) infused with vehicle and losartan for 21 days.\nSaline AngII\nGroups (♂)\nVehicle Vehicle Amlodipine\nNo. N=10 N=10 N=10\nWeight -7 days (g) 35.21 ± 0.48 33.94 ± 0.23 33.51 ± 0.49\nWeight 0 day (g) 35.42 ± 0.51 33.59 ± 0.36 33.54 ± 0.538\nWeight 7 days (g) 33.04 ± 0.57 31.48 ± 0.39 31.74 ± 0.40\nWeight 14 days (g) 33.33 ± 0.31 31.77 ± 0.22 32.26 ± 0.45\nWeight 21 days (g) 34.34 ± 0.63 32.54 ± 0.65 33.41 ± 0.27\nTC (mg/dl) 182.07 ± 7.39 194.53 ± 13.24 190.02 ± 11.27ns\nTG (mg/dl) 90.12 ± 6.65 91.35 ± 7.81 93.14 ± 9.27ns\nEach group weight was compared using Kruskal-Wallis test with Dunn's multiple comparisons test.\nComparison of AngII + Losartan group vs. AngII + Vehicle group.\nFor TC and TG, Kruskal-Wallis test with Dunn's multiple comparisons test.\nData are presented as the mean ± SEM; ns, not significant.\nTC, total cholesterol; TG, triglyceride.\n2. In the Ang II-induced aortic aneurysm model, the authors first induced hypertension in mice and then excluded animals that had\nalready developed aneurysms during the hypertensive phase using ultrasound, followed by initiating CCB intervention. This\nexperimental design mirrors the clinical sequence of antihypertensive treatment in hypertensive patients. However, it is noted that after\nexclusion, no aneurysms were observed in the Ang II + vehicle group, this may lead to misunderstanding. The authors should clarify\nthe exclusion criteria and the number of animals excluded in the manuscript.\nReply: We thank the reviewer for acknowledging the rationale of our animal model design and the thoughtful suggestions. In this\nstudy, 70 sixteen-week-old male C57BL/6J mice were initially infused with AngII for 7 days to induce hypertension. During this period,\n11 mice either died of aneurysm rupture or were diagnosed with aortic aneurysm via ultrasound. The remaining 59 hypertensive mice\nwere subsequently assigned randomly to receive vehicle or CCB treatment (Response Figure 2a). To better illustrate the aortic\npathological changes in the AngII+Vehicle group, we have provided images of all aortas (Response Figure 2b). Although no aortic\naneurysm was observed in the AngII+Vehicle group, the aortas showed obvious expansion compared to those in the Saline+Vehicle\ngroup. Additionally, the diameters of both thoracic and abdominal aortas were significantly increased (red area) (Response Figure 2c–\nf). Furthermore, EVG staining revealed elastic fiber fragmentation in both thoracic and abdominal aortas of the AngII+Vehicle group\n(Response Figure 2g). We have included this information in Extended Data Figure 1a of the revised version. As suggested by the\nreviewer, we have revised the Methods and Results sections in the revised manuscript to avoid misunderstanding: Methods section\n“During hypertension establishment phase, (1) mice died due to aortic aneurysm rupture; or (2) mice diagnosed with developed aorta\naneurysm by ultrasound imaging (TAA or AAA: defined as a segmental aortic diameter > 1.5 times that of the adjacent normal aorta)\nwere excluded from the study and those hypertensive mice without AAD were remained. Subsequently, those hypertensive mice without\nAAD were daily injected with vehicle or one of four CCBs: amlodipine, nifedipine, diltiazem or verapamil”, please refer to Page 41-\n42, Line 896-902. Results section “Sixteen-week-old C57BL/6J mice were subcutaneously infused with AngII (1000 ng/kg·min-1) via\nminipumps for a total of 28 days. First, 70 sixteen-week-old male C57BL/6J mice were initially infused with AngII for 7 days to induce\nhypertension. During this period, 11 mice either died of aneurysm rupture or were diagnosed with aortic aneurysm via ultrasound. Then,\nthe remaining 59 hypertensive mice were randomly assigned to receive either a vehicle or one of four CCBs: amlodipine (1 mg/kg/day),\nnifedipine (20 mg/kg/day), diltiazem (8 mg/kg/day) or verapamil (12 mg/kg/day)”, please refer to Page 7, Line 138-145.\nResponse Figure 2. Design and results-related details of the AngII-induced aortic aneurysm model.\na, Flow chart of the AngII model. Seventy sixteen-week-old male C57BL/6J mice were initially infused with AngII for 7 days to\ninduce hypertension. During this period, 11 mice either died of aneurysm rupture or were diagnosed with aortic aneurysm via\nultrasound. The remaining 59 hypertensive mice were subsequently assigned randomly to receive vehicle or CCB treatment.\nb, Images of all aortas in AngII-induced mouse model. The sacrifice day was indicated below each aorta (day 21 is the endpoint of the\nmodel).\nc-f, Maximal external diameters of ascending aorta, aortic arch, descending aorta and abdominal aorta. The red area represents the\ncomparison between AngII + vehicle and saline + vehicle groups.\ng, Representative images of thoracic and abdominal aorta elastin Van Gieson staining in AngII + vehicle and saline + vehicle groups.\nScale bar=250/50 μm.\nc-f, Data are the mean ± SEM of each group. *P < 0.05; ns, no significant. c-f, Brown-Forsythe and Welch ANOVA test with Dunnett\nT3 multiple comparisons.\nAngII, angiotensin II.\n3. The authors conducted a secondary analysis of a multicenter clinical study and found that CCB use was associated with\nworsened aortic regression/shrinkage in postoperative TBAD patients. However, how should “worsened aortic regression/shrinkage” be\ninterpreted? Does this phenomenon stem from reduced vascular tone caused by CCBs, or does it reflect more adverse pathological\nremodeling of the aorta? The authors did not provide sufficient mechanistic explanations or clinical implications. It is recommended\nthat the authors elaborate on this point in the Discussion section, particularly in the context of postoperative aortic remodeling.\nReply: We appreciate the reviewer’s constructive feedback. Clinical outcomes typically require more than 5 years of follow-up to\nbe observed. Given that the TBAD cohort only has 2 years of follow-up data and the number of clinical outcome events is too small,\nstatistical analysis is not feasible (Response Table 2). As outlined in guidelines from the SVS/STS13, over 60% of type B aortic dissection\npatients experience aneurysmal progression within five years, regardless of initial management. Aneurysmal shrinkage—termed “aortic\nregression/shrinkage” in our study—is recognized by ESC guidelines as a key indicator of low-risk aneurysms during postoperative\nsurveillance14, linked to improved 5–10-year survival and reduced secondary interventions15-18. These rationales guided our choice of\naortic regression/shrinkage as an outcome metric (Response Figure 3). Here, in TBAD patients after TEVAR, we observed a progressive\ndivergence in aortic diameters/volumes over time between the CCB and non-CCB groups during the 2-year follow-up period (Figure\n6e-f). This change is the result of long-term postoperative structural remodeling of the aorta (an elastic artery), rather than rapid changes\nin vascular tone. Vascular tone changes primarily involve alterations in the diameter of small-to-medium arteries19,20. In response to the\nreviewer’s suggestion, we have incorporated additional discussion regarding postoperative aortic remodeling in the revised manuscript:\n“In addition, we selected postoperative aortic regression/shrinkage as the primary outcome, which is endorsed by the ESC guidelines\nas a key indicator of low-risk aneurysms during postoperative surveillance and is associated with improved 5~10-year survival as well\nas reduced rates of secondary interventions. It should be emphasized that this aortic regression/shrinkage does not reflect an acute\nalteration in vascular tone induced by antihypertensive therapy, as we observed a progressive divergence in aortic diameters and volumes\nover time between the CCB and non-CCB groups during the 2-year follow-up period. Therefore, this may reflect CCB-induced\npostoperative aortic remodeling, highlighting the importance of careful selection of antihypertensive therapy in this population and\nunderscoring the urgent need for safer and more effective strategies for both AAD prevention and postoperative management.”. Please\nrefer to Page 17, Line 397-407.\nResponse Table 2. Clinical outcomes between CCBs use and other antihypertensive medication\nOther antihypertensive\nCCBs use\nNumber of patients medication P value\n(n=82)\n(n=28)\nAll-cause death 5 (6.1%) 0 0.223\nStroke 2 (2.4%) 2 (7.4%) 0.268\nRespiratory failure 1 (1.2%) 1 (3.6%) 0.446\nRTAD 1 (1.2%) 0 0.745\nRe-intervention 3 (3.7%) 1 (3.6%) 0.732\nSpinal cord ischemia 1 (1.2%) 0 0.745\nStent stenosis or occlusion 1 (1.2%) 0 0.745\nStent migration 1 (1.2%) 0 0.745\nSINE 0 1 (3.6%) 0.255\nType I Endoleak 5 (6.1%) 0 0.223\nRe-included the previously excluded patients (except for non-hypertensive and loss follow-up patients, n=10).\nRTAD, retrograde type A aortic dissection; SINE, stent-induced new entry.\nResponse Figure 3. Schematic diagram of aortic regression/shrinkage after TEVAR\nThe left column shows CTA cross-sectional images at the level just below the pulmonary artery bifurcation; The right column shows\nmagnified images of the aortic region.\nTEVAR, thoracic endovascular aortic repair.\n4. In animal AAD models, CCBs significantly increased aortic stiffness (elevated PWV and reduced aortic strain), which\nappeared to precede aortic dilation. Is this effect a direct action of CCBs or an early change accelerating disease progression? Would\nadministering CCBs to healthy mice produce similar effects? Additionally, the authors should discuss the potential mechanisms underlying CCB-\ninduced early changes in vascular stiffness.\nReply: We sincerely thank the reviewer for the insightful comment. To explore whether CCBs directly affect aortic stiffness, 16-\nweek-old C57BL/6J mice were administered amlodipine (1 mg/kg/day) for 14 days, and aortic ultrasound assessments were performed\non days 0, 3, 7, and 14 (Response Figure 4a). The results showed that amlodipine did not reduce blood pressure in normotensive mice\n(Response Figure 4b), which is consistent with previous findings in both healthy humans and mice21-23. No significant differences were\nobserved in thoracic or abdominal aortic diameters, nor in rd-PWV and aortic strain between the amlodipine-treated group and the\nvehicle control group (Response Figure 4c-k). These findings suggest that CCBs do not directly cause aortic stiffness or induce aortic\npathology.\nThe stiffness of the aorta is jointly determined by its extracellular matrix and the contractile/relaxant functions of VSMCs. Elastic\nfibers provide the vessel with elastic recoil capacity, while rigid collagen fibers prevent excessive vascular dilation. VSMCs sense\nmechanical pressure changes caused by regular blood pumping and respond adaptively through contraction and relaxation, thereby\nregulating vascular compliance. In the current study, we observed that in AngII- and BAPN-induced AAD model mice, CCB treatment\nled to increased vascular stiffness prior to aortic aneurysm occurrence. We proposed several potential mechanisms: (1) When CCBs are\nadministered to AAD model mice, sustained inhibition of VSMC contraction directly results in the loss of VSMCs’ regulatory effect\non aortic compliance, leading to increased vascular stiffness. Subsequently, impaired mechanical sensing promotes the development of\naortic aneurysms. This hypothesis is supported by the fact that numerous structural mutations associated with aortic disease converge\non the “elastin-contractile-unit”, which refers to the mechano-transduction pathway of VSMC24,25. (2) Under pathological conditions,\nCCB-mediated inhibition of calcium signaling may accelerate the phenotypic switching of VSMCs to a synthetic phenotype26,\npromoting collagen (particularly type I collagen) synthesis and deposition, which directly increases vascular stiffness; (3) CCBs may\nalso inhibit calcium-dependent eNOS activity in endothelial cells, reducing nitric oxide (NO) production26. Insufficient NO weakens its\nregulatory effect on vascular smooth muscle relaxation, leading to reduced vascular elastic reserve and indirectly manifesting as\nincreased stiffness. Regarding which mechanism plays a dominant role in this process, further exploration in subsequent studies may\nbe required. As suggested by the reviewer, we have added the following discussion: “The potential mechanism by which CCBs increase\naortic stiffness include directly inhibiting the contraction of VSMCs and thus impairing their ability to regulate aortic compliance,\npromoting the phenotypic switching of VSMCs to a synthetic phenotype to enhance collagen secretion and deposition, and inhibiting\nthe calcium-dependent endothelial nitric oxide synthase (eNOS) activity in endothelial cells to reduce NO production.”. Please refer to\npage 16, line 375-379.\nResponse Figure 4. CCBs do not directly cause aortic stiffness or induce aortic pathology.\na, Flow chart of the CCB alone model. 16-week-old C57BL/6J mice were administered amlodipine (1 mg/kg/day) for 14 days, and\naortic ultrasound assessments were performed on days 0, 3, 7, and 14. saline + vehicle (n=5), saline + vehicle (n=4).\nb, The SBP of mice were recorded by tail-cuff blood pressure measurement at 0, 3,7,14 days after vehicle/CCBs treatment.\nc, Representative ultrasound images of thoracic and abdominal aorta after 14 days of vehicle/amlodipine treatment.\nd-h, Ultrasound-monitored maximal inner diameters of the aortic root, ascending aorta, aortic arch, descending aorta and abdominal\naorta in mice at different time points.\ni-k, Monitoring of rd-PWV, ascending aortic and abdominal strain in mice at different time points by M-mode ultrasound.\nb, d-k, Data are the mean ± SEM of each group. *P < 0.05; ns, no significant. c-f, Two-way ANOVA with Tukey multiple\ncomparisons test.\nAngII, angiotensin II; SBP, systolic blood pressure; CCB, calcium channel blocker; rd-PWV, root to descending aorta pulse wave\nvelocity.\n5. Please provide sex and gender for human and mice studies in the Method or Result section. Does the sex affect CCBs-induced\nAAD?\nReply: We sincerely thank the reviewer for the insightful suggestion. To explore whether sex may influence CCB-induced AAD,\nwe further analyzed the association of CCB use and incident AAD in males and females among hypertensive participants receiving\nantihypertensive medications in the UKB dataset. We observed that the overall incidence of AAD was higher in males, approximately\nthree times that in females (Response Table 3). Compared with participants using other antihypertensive medications, CCB use appeared\nto increase the incidence of AAD in both sexes, with a potentially greater effect in females. As suggested by the reviewer, we have\nadded this data to Extended Data Table 7 and Result section: “To minimize the indication bias that medication use itself might reflect\ndisease severity, we restricted the analyses to the participants receiving antihypertensive medications and observed that CCB users still\nhad a significantly greater risk of AAD (HR=1.23, 95% CI: 1.09-1.38) than other antihypertensive medication users, with consistent\nresults in both males and females and a relatively greater effect observed in females”. Please refer to page 6-7, line 115-120.\nResponse Table 3. Stratified analysis of sex on the association of CCBs [CCBs vs other antihypertensive medications] and\nAAD among UKB participants with hypertension and antihypertensive medications use (n=98882) a\nStratifies No. cases/person years Hazard ratio (95% CI) a P for interaction\nMen 601/249,316 1.15 (1.04, 1.28) 0.02\nFemale 155/180,371 1.53 (1.25, 1.89)\na Cox model was adjusted for age (years), BMI (kg/m2), total cholesterol (mmol/L), triglycerides (mmol/L), smoking status (never;\nprevious; current), education (College or University degree; A levels or equivalent; O levels or equivalent; None of the above),\nethnicity (white; or not), and systolic blood pressure (mmHg).\nAAD, aortic aneurysm and dissection; CCB, calcium channel blocker.\nMinor Comments\n1. In lines 63-64, the authors state that “there are no currently proven drugs to slow aortic expansion or prevent aortic\nrupture in AAD patients (except for Marfan Syndrome).” While β-blockers or ARBs are recommended for patients with Marfan\nsyndrome, not all studies consistently support the efficacy of these therapies. Therefore, this statement is not entirely accurate and should be revised\nto reflect the variability in current evidence.\nReply: We thank the reviewer for this careful reminder. Although early studies reported that losartan did not significantly limit\naortic dilatation in Marfan syndrome over a 3-year period4, subsequent long-term RCT studies have shown that losartan reduces the rate\nof aortic arch dilatation compared with placebo. Additionally, the combination of ARBs and β-blockers has been associated with a\ndecrease in adverse clinical outcomes5. Accordingly, the 2022 ACC/AHA and 2024 ESC guidelines recommend β-blockers or ARBs\nfor the management of Marfan syndrome to reduce aortic dilatation (Class I, Level A), and support the combination therapy of β-\nblockers and ARBs as Class IIa, Level A14,27. However, for non-hereditary aortic diseases—including thoracic aortic dissection (TAD),\nthoracic aortic aneurysm (TAA), and abdominal aortic aneurysm (AAA)—most pharmacologic studies have only been conducted in\npatients with small AAAs (As summarized in Response Table 4). To date, no pharmacological agent has been proven effective in\nslowing AAA progression, while for TAD and TAA, there are currently no RCTs evaluating pharmacologic interventions. To avoid\nmisunderstanding, we have revised the original sentence to: “at present, only β-blockers or angiotensin II receptor blockers (ARBs) are\nrecommended for aortic aneurysms in patients with Marfan syndrome. There are currently no proven drugs to prevent the occurrence\nof or slow the expansion of the aorta in patients with non-hereditary AAD”. Please refer to page 4, line 63-66.\nResponse Table 4. Summary of the RCT studies regarding drugs and abdominal aortic aneurysm\nFollow-up\nStudy Type Year Journal No. Group Conclusion\n(year)\nPemirolast vs. Do not reduce\nRCT 2015 Br J Surg28 326 1\nPlacebo growth rates\nPerindopril vs.\n73 vs. 72 vs. Do not reduce\nRCT 2016 Eur Heart J1 Amlodipine 2\n79 growth rates\nvs. Placebo\nJ Am Heart Fenofibrate vs. Do not reduce\nRCT 2018 70 vs. 70 0.5\nAssoc29 Placebo growth rates\nJAMA Telmisartan Do not reduce\nRCT 2020 107 vs. 103 2\nCardiol2 vs. Placebo growth rates\nDoxycycline Do not reduce\nRCT 2020 JAMA30 129 vs. 125 2\nvs. Placebo growth rates\nCardiovasc Ticagrelor vs. Do not reduce\nRCT 2020 72 vs. 72 1\nRes31 Placebo growth rates\n2. How was the dosage of CCBs determined in animal models? The manuscript would benefit from a more detailed description of\nthe dosing strategy in the Methods section.\nReply: We thank the reviewer for the question. We used standard interspecies dose conversion based on body surface area. As\noutlined in the FDA’s guidance for dose translation (https://www.fda.gov/media/72309/download), the established conversion factor\nfrom mice to humans is 12.3, which we strictly applied in our study. The corrected human equivalent doses, calculated using this\nframework, are detailed in Response Table 5. As suggested by the reviewer, we have added more detailed descriptions of drug dosages\nin the Methods section. Please refer to page 41, line 884-886.\nResponse Table 5. Summary of mouse-to-human (for 60kg/person) drug dose conversion used in the study\nMouse doses Human doses Human total doses FDA MRHD\nCCBs\n(mg/kg/day) (mg/kg/day) (mg/day) (mg/day)\nAmlodipine 1 0.08 4.8 <10\nNifedipine 20 1.62 97.2 <120\nDiltiazem 8 0.65 39 <480\nVerapamil 12 1 60 <480\nMRHD, maximum recommended human dose.\n3. In Figure 5, the representative EVG staining of the BAPN+Vehicle group does not appear to show substantial elastic fiber\nfragmentation or degradation. To enhance the reliability of histological interpretation, it is recommended that the authors provide\nadditional EVG-stained images for this group.\nReply: We thank the reviewer for this comment. As previously noted, 7/15 mice in the BAPN control group developed TAAD.\nFurther EVG staining showed the average grade of elastic fiber rupture in this group was approximately grade 2, indicating 25–50%\nelastic fiber fragmentation (Extended Data Figure 3d). In Response Figure 5, we showed all aorta of BAPN-control mice and EVG\nstaining from 8 BAPN-control mice, with arrows highlighting varying degrees of aortic pathological changes. As suggested by the\nreviewer, we have changed the representative image of EVG staining of the BAPN+Vehicle group in Figure 5e.\nResponse Figure 5. Results-related details of the BAPN-induced TAAD model.\na, Images of all BAPN + vehicle group aortas in BAPN-induced mouse model. The sacrifice day was indicated below each aorta (day\n21 is the endpoint of the model).\nb, EVG staining from 8 BAPN + vehicle mice, with arrows highlighting varying degrees of aortic pathological changes.\n4. In Extended Data Figure 6, the summary of antihypertensive use in the post-surgery phase could be expanded. Please provide\nmore details and discuss postoperative hypertensive medication in the Discussion section.\nReply: We appreciate the reviewer’s suggestion. According to the 2022 AHA and 2024 ESC guidelines, only β-blockers are\nrecommended for long-term use after aortic dissection surgery to control heart rate and blood pressure (without an assigned level of\nevidence). There is no clear statement regarding the postoperative use of antihypertensive drugs for TAA or AAA. Therefore, we\nsummarized the available clinical studies on the association between postoperative antihypertensive therapy and prognosis in AAD\npatients (Response Table 6), including a total of 13 studies: 1 RCT, 1 case-control study, and 11 retrospective studies. None of these\nstudies were included in the 2022 AHA or 2024 ESC guidelines. Their results were inconsistent and disorganized, with several key\nlimitations: (1) patients undergoing different surgical techniques were analyzed together; (2) patients undergoing the same techniques\nbut using different devices were analyzed together; (3) lesions at different anatomical sites were also analyzed together. These factors\nreduced the accuracy of the findings. In contrast, in the postoperative analysis of our study, all patients underwent TEVAR with the\nsame stent-graft type (T-BranchTM) and standardized surgical procedures, thereby minimizing procedural variability and allowing the\npharmacological effects to be more accurately evaluated. We summarized the studies on antihypertensive drug use after AAD surgery\nin Response Figure 6 (previously Extended Data Figure 6). We have also briefly addressed this point in the revised Discussion section:\n“Current guidelines do not provide specific recommendations regarding antihypertensive therapy after AAD surgery, except for\nsuggesting long-term use of β-blockers after aortic dissection to control heart rate and blood pressure (without an assigned level of\nevidence). To date, only a dozen retrospective studies have investigated the association between antihypertensive medications and\noutcomes after aortic repair, but the findings remain inconsistent. The main reason is that these studies differed substantially in patient\npopulations, study methodologies, surgical approaches (endovascular versus open repair), operative techniques, and graft materials used.\nIn our study, all patients had TBAD and underwent TEVAR with the same stent graft system and a standardized implantation protocol,\nwhich substantially minimized potential confounding factors.”, please refer to page 17, line 387-397.\nResponse Table 6. Summary of the clinical studies regarding hypertensive medication use in AAD post-surgery phase\nSurgical\nStudy Type Year Journal No. Group Conclusion OR/HR\nmodality\n227\nabdominal metoprolol vs. Do not reduce 6-month\nRCT 2006 Am Heart J32 OAR 0.73 (0.23, 2.39)\naortic placebo cardiovascular deaths\naneurysms\n178\nabdominal CCB vs. non- Increased risk of\nCase control 2022 Int Angiol33 EVAR 2.11 (1.05-4.25)\naortic CCB type II endoleak↑\naneurysms\nIncreased 30-day\nDHP-CCB 2.5 (1.3, 4.6)\nmortality↑\nBr J 1000 aortic non-DHP-\nAnaesth34 aneurysms CCB\nDecreased 30-day\nβ-Blocker 0.5 (0.3, 0.9)\nmortality↓\nAnaesth35 aortic non-RAASI mortality↑\naneurysms\n112\nabdominal CCB vs. non- reduce AAA sac\naortic CCB regression↓\naneurysms\n198\nβ-Blocker vs.\nabdominal Do not reduce AAA sac\naortic regression\nBlocker\naneurysms\n6515\nβ-Blocker vs.\nabdominal Reduced postoperative\nRetrospective aortic mortality↓\nBlocker\naneurysms\n4275 acute\nJ Thorac β-Blocker vs.\ntype A Reduced major adverse\naortic cardiac events↓\nSurg39 Blocker\ndissection\n1752 type β-Blocker vs.\nAnn Vasc TEVAR or Reduced mortality and 0.53 (0.32, 0.90)\nSurg40 OAR stroke↓ 0.46 (0.25, 0.87)\ndissection Blocker\n1114 type β-Blocker vs.\nPromote false lumen\nthrombosis\ndissection Blocker\n1821 type\nA and 1172 β-\nFront TEVAR or Reduced type B\n2023 type B Blocker+CCB -\nPharmacol42 OAR composite outcomes↓\naortic vs.β-Blocker\ndissection\nJ Am Heart 7106 aortic EVAR or RAASIs vs. Reduced 1-year 0.45 (0.4, 0.5)\n50411 β-Blocker vs.\nAnn Vasc Increased 1-year\n2025 aortic EVAR non-β- 1.26 (1.10, 1.41)\nSurg44 mortality↑\naneurysms Blocker\nOAR, open aortic repair; EVAR, endovascular aortic repair; TEVAR, thoracic endovascular aortic repair; DHP, dihydropyridines;\nRAASI, renin-angiotensin-aldosterone system inhibitors.\nResponse Figure 6. Summary of clinical studies on the use of antihypertensive medication across different stages of AAD.\nAAD, aortic aneurysm and dissection; TAA, thoracic aorta aneurysm; AAA, abdominal aorta aneurysm; TAAD, thoracic aorta\naneurysm and dissection; AHA, American Heart Association; ESC, European Society of Cardiology; MFS, Marfan Syndrome; ARB,\nangiotensin II receptor blocker; CCB, calcium channel blocker; ACEI, angiotensin-converting enzyme Inhibitor; TEVAR, thoracic\nendovascular aortic repair; RAASIs, renin-angiotensin-aldosterone system inhibitor.\nReviewer #2:\nThe study under review presents a groundbreaking investigation into the potential association between calcium channel\nblockers (CCBs) and aortic aneurysm and dissection (AAD), two life-threatening cardiovascular conditions. The findings are of paramount\nimportance, given the prevalence of hypertension and the widespread use of CCBs as a first-line treatment. The study's large sample\nsize and long follow-up period lend significant weight to its conclusions, which suggest that CCBs may be linked to an increased risk\nof AAD. This revelation could have far-reaching implications for clinical practice, potentially altering the way clinicians approach the\nmanagement of hypertension in patients at risk of AAD. However, there are several aspects that could be further refined to strengthen\nthe study's conclusions.\nReply: We appreciate the reviewer for the positive feedback.\nFirstly, the research on downstream mechanisms is relatively superficial. The study suggests that CCBs may affect AAD through\ntheir impact on vascular smooth muscle cells (VSMCs). However, more rigorous cell experiments are needed to confirm this. It is\nunclear whether the effect is direct or if there are intermediate factors involved. For instance, CCBs might influence intracellular calcium\nsignaling pathways in VSMCs, which in turn affect cell contraction and proliferation. Detailed molecular studies on VSMCs treated\nwith CCBs could provide valuable insights into the underlying mechanisms.\nReply: We thank the reviewer for the constructive suggestion. Previous study revealed that CCBs exacerbate aortic dilation in\nMarfan Syndrome by activating ERK1/2 signaling45. Here, in AngII-treated VSMCs, we observed that both amlodipine and verapamil\nenhanced ERK1/2 activation and promoted IL-6 and MCP-1 expression. Pharmacological inhibition of ERK1/2 with U0126\nsignificantly blocked CCB-aggravated inflammatory factor expression (Response Figure 7). As suggested by the reviewer, we have\nadded this data to Extended Data Figure 1 in the revised version of the manuscript, please refer to page 9, line 185-191. Next, to\ncomprehensively profile the systemic changes induced by CCBs in VSMCs, we performed RNA sequencing on AngII- and CCB-treated\nVSMCs. As shown in Response Figure 8a, VSMCs were treated with AngII (1 μM) together with Vehicle, amlodipine (20 nM) or\nverapamil (0.5 μM) for 24 hours and then subjected to RNAseq. 453 upregulated and 521 downregulated differentially expressed genes\n(DEGs) were identified in amlodipine-treated VSMCs. KEGG analysis showed that the downregulated DEGs were mainly enriched in\ncytoskeleton in muscle cells, focal adhesion, and protein digestion and absorption pathways, while the upregulated DEGs were primarily\nenriched in pathways related to herpes simplex virus 1 infection, Epstein-Barr virus infection, and Influenza A (Response Figure 8b-d).\nIn verapamil-treated VSMCs, 401 DEGs were upregulated and 471 were downregulated. The downregulated DEGs were mainly\nenriched in ECM-receptor interaction, AGE-RAGE signaling pathway, and pathways in cancer, whereas the upregulated DEGs were\nenriched in ribosome, coronavirus disease, and microRNAs in cancer pathways (Response Figure 8e-g). Both amlodipine and verapamil\ndownregulated genes related to focal adhesion and ECM-receptor interaction, which is consistent with the observation that numerous\nstructural mutations associated with aortic disease converge on the “elastin-contractile unit”24,25. However, confirming this mechanism\nrequires extensive further investigation, which is beyond the scope of the present study. We plan to pursue this in our future work.\nResponse Figure 7. Amlodipine and verapamil enhanced ERK1/2 activation and promoted IL-6 and MCP-1 expression.\na, Representative western blot image and statistical analysis of ERK1/2 phosphorylation in VSMC stimulated with AngII (1 μM) for\n12h followed by vehicle, amlodipine (20 nM) and verapamil (0.5 μM) for 24 hours.\nb, Statistical analysis of IL-6 and MCP1 relative mRNA level in VSMC stimulated with AngII (1 μM) + DSMO or AngII (1 μM) +\nU0126 for 12h followed by vehicle, amlodipine (20 nM) and verapamil (0.5 μM) for 24 hours.\na, b, Data are the mean ± SEM of each group. *P < 0.05; ns, no significant. a, One-way ANOVA with Holm-Sidak multiple\ncomparisons test; b, Brown-Forsythe and Welch ANOVA test with Dunnett T3 multiple comparisons.\nAng II, angiotensin II; ERK1/2, extracellular signal-regulated kinases 1 and 2; IL-6, interleukin-6; MCP1, monocyte chemoattractant\nprotein 1; U0126, a specific inhibitor of ERK1/2.\nResponse Figure 8. Both amlodipine and verapamil downregulated genes related to focal adhesion and ECM-receptor\ninteraction.\na, Schematic of the VSMC intervention workflow: VSMCs were serum-starved for 12 hours, followed by AngII (1 μM) stimulation\nfor 12 hours, then treated with amlodipine (20 nM) and verapamil (0.5 μM) for 24 hours. VSMCs were subsequently harvested for\nRNA extraction and RNA sequencing\nb-d, Volcano plot and KEGG pathway analysis of differentially expressed genes between AngII + amlodipine and AngII + vehicle\ngroups. |log FoldChange| > 0 and p < 0.05.\n2\ne-g, Volcano plot and KEGG pathway analysis of differentially expressed genes between AngII + verapamil and AngII + vehicle\ngroups. |log FoldChange| > 0 and p < 0.05.\n2\nh-i, Heatmaps showing molecules related to focal adhesion and ECM-receptor interaction that were downregulated by amlodipine and\nverapamil, respectively.\nDEG, differentially expressed genes.\nSecondly, it would be beneficial to investigate whether there is any known relationship between the calcium channels in\nVSMCs and PRKG1. PRKG1 has been shown to restore VSMC contractility and alleviate CCB-induced aortic stiffness and\nAAD incidence. Understanding the potential crosstalk between these two pathways could shed light on the complex\ninteractions within the vascular wall and help identify new therapeutic targets.\nReply: We thank the reviewer for the valuable suggestion regarding the relationship between calcium channels and PRKG1.\nVSMC contraction is central to vascular tone regulation, with calcium signaling acting as the \"molecular switch\" that initiates this\nprocess. Upon contractile stimulation-induced membrane depolarization in VSMCs, L-type calcium channels open, enabling massive\nextracellular Ca²⁺ influx. Elevated intracellular Ca²⁺ binds to ryanodine receptors, triggering further Ca²⁺ release from endoplasmic\nreticulum stores to amplify calcium signaling. Intracellular Ca²⁺ then binds to calmodulin (CaM) to form a complex, which activates\nmyosin light chain kinase (MLCK) and leads to the phosphorylation of myosin light chain (MLC). Phosphorylated myosin binds to\nactin filaments to form “cross-bridges” and generates contraction. PRKG1, a key effector molecule in the NO-cGMP pathway, can\nsignificantly enhance the activity of myosin light chain phosphatase (MLCP) by phosphorylating MYPT1, thereby inhibiting MLC\nphosphorylation and VSMC contraction. Of note, PRKG1 can directly phosphorylate the α1c subunit of the L-type calcium channel\nCaV1.2 (e.g., at the Ser1928 site), suppressing extracellular Ca²⁺ influx46. In the present study, given that PRKG1 is a key endogenous\ninhibitor of the calcium-CaM-MLC signaling pathway, and its gain-of-function mutations have been confirmed to cause hereditary\nthoracic aortic aneurysm and dissection (TAAD), we enhanced calcium-MLC signaling in VSMCs of CCB-treated mice by knocking\ndown PRKG1. We found that the development of AAD induced by CCBs was significantly attenuated. These results confirm that CCBs\ninfluence the development of AAD by directly affecting the calcium-MLC-contraction pathway. Here, when we treated VSMCs with\nCCBs to inhibit calcium signal, we did not observe a significant change in PRKG1 expression (Response Figure 9a). Therefore, CCBs\ncould influence the PRKG1 activity, as reflected by downstream MYPT1 phosphorylation (Response Figure 9b). Following the\nreviewer’s suggestion, we added a discussion on the interaction between calcium channels and PRKG1 in the manuscript: “In contrast,\nin vivo knockdown of PRKG1, which is an endogenous inhibitor of MLC phosphorylation and can directly phosphorylate the α1c\nsubunit of the L-type calcium channel CaV1.2 (e.g., at the Ser1928 site), suppressing Ca²⁺ influx, restored VSMCs contractility and\nnotably mitigated the CCB-induced exacerbation of AAD.”. Please refer to page 16, line 363-366.\nResponse Figure 9. CCBs did not affect PRKG1 protein expression in VSMCs, but promoted phosphorylation of its\ndownstream target MYPT1.\na, Representative western blot image and statistical analysis of Prkg1 protein level and mRNA level in VSMC stimulated with AngII\n(1 μM) for 12h followed by vehicle, amlodipine (20 nM) and verapamil (0.5 μM) for 24 hours.\nb, Diagram of the signaling involved in VSMC contraction. And representative western blot image and statistical analysis of MLC\nphosphorylation, and MYPT1 phosphorylation in aortas from mice infused with AngII (1000 ng/kg·min-1) for 7 days or treated with\n0.3% BAPN for 3 days followed by injection of vehicle or verapamil (20 mg/kg/d) for 1 hour.\na, b, Data are the mean ± SEM of each group. *P < 0.05; ns, no significant. a, Brown-Forsythe and Welch ANOVA test with Dunnett\nT3 multiple comparisons. b, Unpaired Student T test.\nLTCC, L-type calcium channel; MLC, myosin regulatory light chain; MLCK, myosin light chain kinase; PRKG1, cGMP-dependent\nprotein kinase type 1; MLCP, myosin light chain phosphatase; MYPT1, myosin phosphatase target subunit 1.\nThirdly, there are some studies that have shown CCBs may alleviate aortic aneurysm and dissection (J Mol Cell Cardiol. 2015\nAug 4;87:152–159; Front Pharmacol. 2023 Nov 10;14:1291900). The article should discuss these conflicting conclusions and explore\nthe possible reasons. Differences in study populations, methodologies, and definitions of AAD could contribute to the divergent results.\nA thorough comparison and analysis of these studies would provide a more comprehensive understanding of the role of CCBs in AAD\nand help resolve the inconsistencies in the literature.\nReply: We appreciate the reviewer for the suggestion. The reviewer cited two representative studies suggesting that CCBs may\nalleviate aortic aneurysm and dissection in animal experiments (J Mol Cell Cardiol. 2015 Oct;87:152-9) and clinical research (Front\nPharmacol. 2023 Nov 10;14:1291900). As noted by the reviewer, these conflicting conclusions reflect the complexity of the issue. Here,\nwe systematically dissect the underlying factors involved and provide reasonable explanations.\nFirstly, regarding animal studies on CCBs and AAD (summarized in Response Table 7), although several studies have reported\nthat CCBs may alleviate AngII-induced aortic aneurysms in mice, these studies have two major limitations: (1) In all AngII models,\nAngII and CCBs were administered simultaneously. This resulted in the failure to establish a valid AngII-induced hypertension model\nin CCB-treated mice, which fails to simulate real-world clinical scenarios of CCB use. (2) Most studies used supratherapeutic doses of\nCCBs—even 5–10 times higher than the maximum clinical dose47-49. Such experimental design raises the possibility that the observed\nprotective effects are artificial. In contrast, a 2015 study by Harry Dietz et al.45 administered CCBs to Marfan mice with Fbn1 mutations\nand observed a significant increase in aortic diameter—a finding cited in the AHA/ESC guidelines. To address these, here, we\nadministered various CCBs at clinical doses to mice after successfully establishing a hypertension model. Our results showed that CCBs\nsignificantly increased the incidence of AAD and aortic diameter, which is consistent with the conclusions from our analysis of the\nlarge-scale general population cohort in the UKB.\nSecondly, regarding clinical studies on CCBs and AAD (summarized in Response Table 8), as noted by the reviewer, these studies\nreported conflicting results. Given that patients may receive CCBs at different stages of AAD, and that aortic aneurysm and aortic\ndissection have distinct characteristics, we categorized these 11 studies according to the disease stage. Of these, 7 studies focused on\nestablished AAD, and 4 studies on AAD after surgery. However, no study has investigated CCB use and the risk of new-onset AAD in\nhypertensive patients. Among the 7 studies on established AAD, patients with aortic dissection, showing that conservative blood\npressure control with CCBs could improve prognosis—consistent with the AHA/ESC guideline recommendation to lower blood\npressure to prevent further tearing50-53. In contrast, for patients with aortic aneurysm, CCB use showed no significant delaying effect1\nand, in some studies, even a worsening effect45,54. In studies on CCB use and postoperative AAD prognosis, conclusions were\ninconsistent, largely due to heterogeneity in disease inclusion, surgical procedures, and device types (described in Response Figure 6).\nFollowing the reviewers’ suggestions, this has been discussed in the “Discussion” section, please refer to page 14 line 310-324;\npage 15 line 348-351; page 17 line 387-407.\nResponse Table 7. Summary of the animal studies regarding CCBs and AAD\nAAD or\nYear Journal Animal Model Conclusion\ndiameter\n350-400g male Azelnidipine (2 mg/kg/d) Reduced maximal Diameter (mm)\nWistar rats prior to Elastase diameter↓ 10.7 vs. 7.9\nCo-infused\nmale Reduced maximal Diameter (mm)\nWistar rats diameter↓ 3.28 vs. 2.41\nElastase\nCo-infused\nIncidence (%)\n9-week male Amlodipine (5 mg/kg/d) Reduced aortic\nC57 mouse AngII (1000 aneurysms↓\nAAA: 49 vs. 0\nng/kg/d)+BAPN\nCo-infused\n8-week male Reduced aortic Incidence (%)\nAPOE-/- mouse aneurysms↓ 60 vs. 0\nAngII (1440 ng/kg/min)\nCo-infused\nMale LDLR-/- Reduced maximal Diameter (mm)\nmouse diameter↓ 1.72 vs. 1.02\nAngII (1000 ng/kg/min)\nCo-infused\nJ Thorac Azelnidipine (0.5\n7-week male Incidence (%)\nC57 mouse 91 vs. 18\nSurg57 AngII (1000\nng/kg/d)+BAPN\nAmlodipine (12 Increased\n2-month marfan\nmouse\nVerapamil (20 mg/kg/d) expansion↑\nCo-infused\nJ Mol Cell 6-month male Nifedipine (5, 20 Incidence (%)\nCardiol58 hph1-/- mouse mg/kg/d) and AngII (700 71 vs. 7\nng/kg/min)\nResponse Table 8. Summary of the clinical studies regarding CCBs and different stages of aortic aneurysm and dissection\n1. Hypertension without AAD patients: Association between CCB use and the risk of new-onset AAD\nStudy Type Year Journal No. Group Conclusion OR/HR\nNo study\n2. Established AAD patients: Association between CCB use and aortic aneurysm growth or aortic dissection\nprognosis\nStudy Type Year Journal No. Group Conclusion OR/HR\n72 small AAA Amlodipine Do not reduce AAA\nRCT 2016 Eur Heart J1 -\npatients vs. Placebo growth rates\n5373 patients non-CCB Arterial stiffness↑\nCase control\n1819 others non-CCB\n202 acute B CCB vs. No\nAm J\n2009 dissection hypertensive All-cause mortality↓ 0.38\nHypertens50\npatients medication\n191 acute B\nAnn Thorac\n2012 dissection CCB Aortic expansion rate↓ -3.85\nSurg51\npatients\n722 type A Survival of Type B with\nRetrospective Am J dissection and medication↑ 0.55\nCardiol52 579 type B Survival of Type A with 1.48\ndissection surgery↓\n3932 non-\nβ-blocker Risk of the composite\n+CCB outcome↓\npatients\n3. AAD patients after surgery: Association between CCB use and AAD prognosis\nStudy Type Year Journal No. Group Conclusion OR/HR\nCase control 2022 Int Angiol33 CCB Type II endoleak↑ 2.11\nEVAR patients\nBr J CCB vs.\n2008 surgery Perioperative mortality↑ 2.5\nAnaesth34 non-CCB\npatients\nRetrospective EVAR patients non-CCB\n1821 type A\nCCB+β-\nFront and 1172 type Reduced type B\nPharmacol42 B aortic composite outcomes↓\nvs.β-Blocker\ndissection\nIn conclusion, the study provides valuable insights into the potential relationship between CCBs and AAD, highlighting the need\nfor caution when prescribing CCBs to hypertensive patients at risk of AAD. The findings are particularly important given the widespread\nuse of CCBs in clinical practice. Addressing the aforementioned points through a revision process would significantly enhance the\nrobustness and credibility of the findings.\nReply: We sincerely thank you for your thoughtful and encouraging comments. We have carefully addressed all your suggestions\nto enhance the scientific rigor and overall quality of the revised manuscript.\nReferences\nenzyme inhibitor on the growth rate of small abdominal aortic aneurysms: a randomized placebo-controlled trial\n(AARDVARK). Eur Heart J 37, 3213-3221 (2016). https://doi.org:10.1093/eurheartj/ehw257\nTrial. JAMA Cardiol 5, 1374-1381 (2020). https://doi.org:10.1001/jamacardio.2020.3524\n2071 (2014). https://doi.org:10.1056/NEJMoa1404731\nhttps://doi.org:10.1093/eurheartj/ehv151\n5 van Andel, M. M. et al. Long-term clinical outcomes of losartan in patients with Marfan syndrome: follow-up of the multicentre\nrandomized controlled COMPARE trial. Eur Heart J 41, 4181-4187 (2020). https://doi.org:10.1093/eurheartj/ehaa377\n- a randomised, double-blind, crossover trial. Eur J Clin Invest 42, 891-899 (2012). https://doi.org:10.1111/j.1365-\n2362.2012.02668.x\nenzyme inhibitors. 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Front Pharmacol 14, 1291900 (2023). https://doi.org:10.3389/fphar.2023.1291900\nAssociated With Favorable Outcomes Compared to Beta Blockers in Reducing Mortality Following Abdominal Aneurysm\nRepair. J Am Heart Assoc 12, e029761 (2023). https://doi.org:10.1161/jaha.122.029761\nReinterventions After Endovascular Abdominal Aortic Repair (EVAR). Ann Vasc Surg 110, 395-404 (2025).\nhttps://doi.org:10.1016/j.avsg.2024.07.104\nElife 4 (2015). https://doi.org:10.7554/eLife.08648\nhttps://doi.org:10.1161/circresaha.107.156976\ninflammatory effects. Hypertension 62, 1098-1104 (2013). https://doi.org:10.1161/hypertensionaha.113.01986\n1274 (2010). https://doi.org:10.1161/hypertensionaha.109.140558\nOne 8, e81743 (2013). https://doi.org:10.1371/journal.pone.0081743\n371-377 (2009). https://doi.org:10.1038/ajh.2009.5\nhttps://doi.org:10.1016/j.athoracsur.2012.05.040\nof Acute Aortic Dissection [IRAD]). Am J Cardiol 109, 122-127 (2012). https://doi.org:10.1016/j.amjcard.2011.08.012\nAntihypertensive Drugs in Non-Operated Aortic Dissection Patients. J Clin Med 12 (2023).\nhttps://doi.org:10.3390/jcm12051962\nexperimental abdominal aortic aneurysms. Surg Today 37, 468-473 (2007). https://doi.org:10.1007/s00595-006-3367-6\n(2008).\neffects. J Thorac Cardiovasc Surg 146, 1501-1508 (2013). https://doi.org:10.1016/j.jtcvs.2013.02.073\nmice: Mechanisms and implications. J Mol Cell Cardiol 87, 152-159 (2015). https://doi.org:10.1016/j.yjmcc.2015.07.031\nResponse to reviewers\nWe would like to express our sincere gratitude to the editor and both reviewers for their time and thoughtful evaluation of our\nmanuscript. We truly appreciate their constructive comments and insightful suggestions, which have greatly improved the clarity and\nquality of our work. We are delighted that the reviewers recognize the value and significance of our study, and we are grateful for their\nsupportive feedback.\nReviewer #1:\nnone\nReply: We thank the reviewer for the positive and encouraging feedback. We are pleased that the reviewer finds our revisions\nsatisfactory and has no further concerns. We are grateful for the reviewer’s thoughtful input, which has contributed significantly to\nimproving our manuscript.\nReviewer #2:\nThis manuscript reports an innovative finding that calcium channel blockers (CCBs) may increase the risk of aortic\naneurysm/dissection (AAD) in hypertensive patients, warranting significant attention. This work holds major implications for clinical\nresearch in this field and provides crucial evidence for personalized antihypertensive therapy. Further mechanistic studies are needed\nto elucidate the precise role of PRKG1.\nReply: We sincerely appreciate reviewer thoughtful and supportive comments. We are encouraged that the reviewer recognizes\nthe novelty and clinical importance of our findings that CCBs may increase the risk of AAD in hypertensive patients. We also agree\nwith the reviewer that further mechanistic studies on PRKG1 are warranted to clarify its precise role. We are grateful for this constructive\nperspective, which provides valuable direction for our future research."}, {"round": 2, "reviewer_comments": {"reviewer_2": "This manuscript reports an innovative finding that calcium channel blockers (CCBs) may increase the risk of aortic\naneurysm/dissection (AAD) in hypertensive patients, warranting significant attention. This work holds major implications for\nclinical research in this field and provides crucial evidence for personalized antihypertensive therapy. Further mechanistic\nstudies are needed to elucidate the precise role of PRKG1."}, "author_response": "Response to reviewers\nWe would like to express our sincere gratitude to the editor and both reviewers for their time and thoughtful evaluation of our\nmanuscript. We truly appreciate their constructive comments and insightful suggestions, which have greatly improved the clarity and\nquality of our work. We are delighted that the reviewers recognize the value and significance of our study, and we are grateful for their\nsupportive feedback.\nReviewer #1:\nnone\nReply: We thank the reviewer for the positive and encouraging feedback. We are pleased that the reviewer finds our revisions\nsatisfactory and has no further concerns. We are grateful for the reviewer’s thoughtful input, which has contributed significantly to\nimproving our manuscript.\nReviewer #2:\nThis manuscript reports an innovative finding that calcium channel blockers (CCBs) may increase the risk of aortic\naneurysm/dissection (AAD) in hypertensive patients, warranting significant attention. This work holds major implications for clinical\nresearch in this field and provides crucial evidence for personalized antihypertensive therapy. Further mechanistic studies are needed\nto elucidate the precise role of PRKG1.\nReply: We sincerely appreciate reviewer thoughtful and supportive comments. We are encouraged that the reviewer recognizes\nthe novelty and clinical importance of our findings that CCBs may increase the risk of AAD in hypertensive patients. We also agree\nwith the reviewer that further mechanistic studies on PRKG1 are warranted to clarify its precise role. We are grateful for this constructive\nperspective, which provides valuable direction for our future research."}]

Decision letter