Angiotensin-II (Ang-II) drives pathological vascular wall remodeling in hypertension and abdominal aortic aneurysm (AAA) through mechanisms that are not completely understood. Previous studies showed that the phosphatase activity of calcineurin (Cn) mediates Ang-II-induced AAA, but the cell type involved in the action of Cn in AAA formation remained unknown. Here, by employing newly created smooth muscle cell (SMC)-specific and endothelial cell (EC)-specific Cn-deficient mice (SM-Cn^−/− and EC-Cn^−/− mice), we show that Cn expressed in SMCs, but not ECs, was required for Ang-II-induced AAA. Unexpectedly, SMC Cn also played a structural role in the early onset and maintenance of Ang-II-induced hypertension, independently of its known phosphatase activity. Among the signaling pathways activated by Ang-II, Cn signaling is essential in SMCs, as nearly 90% of the genes regulated by Ang-II in the aorta required Cn expression in SMCs. Cn orchestrated, independently of its enzymatic activity, the induction by Ang-II of a transcriptional program closely related to SMC contractility and hypertension. Cn deletion in SMCs, but not its pharmacological inhibition, impaired the regulation of arterial contractility. Among the genes whose regulation by Ang-II required Cn expression but not its phosphatase activity, we discovered that Serpine1 was critical for Ang-II-induced hypertension. Indeed, pharmacological inhibition of PAI-1, the protein encoded by Serpine1, impaired SMCs contractility and readily regressed hypertension. Mechanistically, Serpine1 induction was mediated by Smad2 activation via the structural role of Cn. These findings uncover an unexpected role for Cn in vascular pathophysiology and highlight PAI-1 as a potential therapeutic target for hypertension.

Smooth muscle Cn is required for AAA formation

To investigate the selective contribution of EC-expressed and VSMC-expressed Cn to pathological VWR, we generated tamoxifen (Tx)-inducible knockout mice conditionally lacking Cn in ECs or smooth muscle cells (SMCs). The catalytic subunit CnA is encoded by 3 genes (Ppp3ca, Ppp3cb, and Ppp3cc), and the regulatory subunit CnB by 2 genes (Ppp3r1 and Ppp3r2). The CnB2 subunit is expressed only in the testes, whereas CnB1 is ubiquitously expressed and is indispensable for maintaining Cn activity [13]. CnA and CnB are obligate heterodimers, such that if CnB is missing, CnA becomes unstable and is degraded [14]. We therefore crossed LoxP-flanked Ppp3r1 (Cnb1^f/f) mice [15] with mice expressing Tx-inducible Cre recombinase (Cre^ERT2) specifically in ECs (Cdh5-Cre^ERT2) [16] or in SMCs (Myh11-Cre^ERT2) [17], obtaining Cnb1^f/f;Cdh5-Cre^ERT2 mice (EC-Cn^f/f) or Cnb1^f/f;Myh11-Cre^ERT2 mice (SM-Cn^f/f), respectively. Cnb1^f/f-Cre^ERT2-neg together with Cnb1^wt/wt;Cdh5-Cre^ERT2 or Cnb1^wt/wt;Myh11-Cre^ERT2, were used as controls (Cn-Ctl) for Cnb1^f/f;Cdh5-Cre^ERT2 or for Cnb1^f/f;Myh11-Cre^ERT2, respectively. Tx treatment induced Cnb1 deletion specifically in ECs (EC-Cn^−/− mice) or SMCs (SM-Cn^−/− mice), as confirmed by immunoblot analysis of CnB expression in mouse aortic endothelial cells (mAECs) from EC-Cn^−/− mice (Fig 1A and 1B) or in aortic extracts from SM-Cn^−/− mice (Fig 1C and 1D). Destabilization of the catalytic subunit CnA in the absence of the regulatory subunit CnB has been demonstrated in vivo [14,18]. As expected, Cnb1 deletion caused a drop in CnA protein expression (Fig 1A–1D). Of note, baseline measurements showed that Cn deficiency in ECs or SMCs did not substantially affect blood pressure (BP), aortic diameter, or aortic tissue organization (Fig 1E–1I).

To assess the contributions of EC-expressed and SMC-expressed Cn to AAA, we used a mouse model [19] involving 4 weeks of Angiotensin II (Ang-II) infusion in mice fed a high-fat diet (HFD) for 12 weeks (Fig 2A). In Cn-Ctl mice, Ang-II treatment induced marked abdominal aorta (AbAo) dilation (Fig 2B–2E) without altering CnA or CnB expression in aortic tissues (S1A and S1B Fig). While Ang-II-induced AbAo dilatation was unaffected by deletion of Cn in ECs, it was abolished by Cn deletion in SMCs (Fig 2B–2E). Cn deletion in ECs or SMCs did not prevent HFD-induced obesity in these mice (S2 Fig). Ex vivo images and histological analysis of AbAo cross sections revealed AbAo remodeling characterized by increased wall thickness, collagen deposition, and elastic fiber disarray in Ang-II-treated Cn-Ctl and EC-Cn^−/− mice that was not present in the AbAo of SM-Cn^−/− mice (Fig 2E–2H). Furthermore, medial layer α-SMA levels were significantly higher in SM-Cn^−/− mice compared to Cn-Ctl and EC-Cn^−/− mice after Ang-II infusion (S3A and S3B Fig). In addition, markers of proliferation (Ki67) and inflammatory cell infiltration were substantially increased in the AbAo of Cn-Ctl and EC-Cn^−/− mice, but not in SM-Cn^−/− mice (S3C–S3F Fig). These findings demonstrate that SMC-expressed Cn, but not EC-expressed Cn, plays a critical role in Ang-II-induced AbAo wall remodeling and AAA formation.

SMC Cn is required for Ang-II-induced hypertension

Ang-II induces vasoconstriction and hypertension in mice, and we therefore investigated whether Cn deficiency affected Ang-II-induced hypertension in the same mice used for the experiments presented in Fig 2. While Cn-Ctl and EC-Cn^−/− mice showed a sharp and sustained increase in systolic and diastolic BP after 1 week of treatment with Ang-II, SM-Cn^−/− mice remained normotensive (Fig 3). These results strongly suggest that SMC Cn is a critical mediator of Ang-II-induced hypertension.

In rabbits, a high-cholesterol diet alters Ang-II signaling in the aorta [20]. Therefore, to exclude possible secondary effects of HFD feeding on BP regulation by SMC Cn, we investigated the contribution of vascular Cn to Ang-II-induced hypertension in mice fed a chow diet. Infusion of Ang-II in Tx-treated mice (Fig 4A) sharply increased BP in Cn-Ctl and EC-Cn^−/− mice from the first week of treatment but failed to substantially raise BP in SM-Cn^−/− mice (Figs 4B, 4C, and S4). Similarly, Ang-II induced AbAo dilatation in Cn-Ctl and EC-Cn^−/− mice from the first week of treatment but did not have this effect in SM-Cn^−/− mice (Fig 4D and 4E). These results support a key role for SMC Cn in Ang-II-induced hypertension and AbAo dilatation.

To investigate whether Cn deletion in SMCs also conferred resistance to hypertension induced by other factors, we treated mice with norepinephrine (NE) or the nitric oxide synthase inhibitor L-NAME (S5A Fig). NE and L-NAME both markedly increased BP in Cn-Ctl mice relative to similarly treated SM-Cn^−/− mice and untreated controls (S5B Fig). These results indicate that SMC-expressed Cn is an essential regulator of BP in response to diverse hypertensive stimuli.

Ang-II-induced hypertension does not require Cn phosphatase activity

A major role for Cn in BP regulation was unexpected because inhibition of Cn activity does not prevent Ang-II-induced hypertension in mice [21] and because long-term treatment of patients with Cn inhibitors causes hypertension, likely as a consequence of nephrotoxicity [22]. A number of factors could account for this discrepancy, including different contributions of Cn to BP regulation in humans and mice, off-target effects of Cn inhibitors, or a distinct effect of pharmacological inhibition versus protein deletion. Pretreatment of mice with the Cn inhibitor CsA did not protect against Ang-II-induced hypertension in WT mice (Fig 4F and 4G); in contrast, CsA pretreatment did block Ang-II-induced AbAo dilatation in these mice (Fig 4H). This result confirms effective phosphatase inhibition and is consistent with a previous report showing that CsA prevents AbAo dilatation and AAA [7]. Demonstrating efficient systemic inhibition of Cn phosphatase activity by CsA, NFATc3, a known Cn substrate, was hyperphosphorylated in the thymus of CsA-treated mice (S6A Fig). Also consistent with previous reports [21], CsA pretreatment significantly reduced Ang-II-induced cardiac hypertrophy in these mice (S6B and S6C Fig). Together, these data indicate that CsA does not prevent Ang-II-induced hypertension and thus suggest that the contribution of Cn to Ang-II-induced hypertension is unrelated to its phosphatase activity.

To confirm the non-involvement of Cn phosphatase activity in Ang-II-induced hypertension, we inhibited Cn activity in vivo by inoculating WT mice with a lentivirus (LV) encoding the LxVP peptide fused to GFP (LV-LxVP). Like CsA, the LxVP peptide blocks the binding of Cn to its substrates, inhibiting its activity [23]. Six weeks after LV inoculation, Ang-II was administered to mice inoculated with LV-LxVP or with LV encoding an inactive mutant version (LV-LxVPmut), and BP was determined (Fig 5A). Ang-II markedly increased systolic and diastolic BP in mice transduced with LV-LxVPmut or LV-LxVP, with no substantial difference between treatment groups (Fig 5B). Efficient transduction of all aortic layers was confirmed by GFP immunostaining of aortic sections (Fig 5C). To assess whether LV-LxVP efficiently blocked Cn/NFAT activation by Ang-II in these mice, we analyzed the subcellular localization and DNA-binding activity of NFAT proteins. NFATs are dephosphorylated by Cn and translocate to the nucleus in active form to bind target gene promoters. In situ southwestern histochemistry detected abundant active NFAT in cell nuclei in medial AbAo and AsAo sections from Ang-II-treated mice expressing LV-LxVPmut, but a nuclear NFAT signal was barely detected in sections from LV-LxVP transduced animals (Fig 5D and 5E). These data further support the idea that SMC Cn mediates Ang-II-induced hypertension independently of its phosphatase activity.

SMC Cn is required for both early onset and long-term sustainability of Ang-II-induced hypertension

To investigate whether smooth muscle Cn was required only for the long-term maintenance of Ang-II-induced hypertension or also for its onset, we measured BP in Cn-Ctl and SM-Cn^−/− mice early after Ang-II infusion (Fig 6A). A robust increase in BP was observed in Cn-Ctl mice as early as 2 h after Ang-II infusion and was maintained after 24 h, whereas SM-Cn^−/− mice remained normotensive (Figs 6B and S7A). In parallel studies, WT mice were treated with CsA for 5 days before Ang-II infusion (Fig 6C). Consistent with the results of the long-term experiment with CsA (Fig 4F and 4G), CsA pre-treatment failed to prevent Ang-II-induced hypertension after 2 or 24 h (Figs 6D and S7B). Efficient inhibition of Cn phosphatase activity in CsA-treated mice was confirmed by the NFAT phosphorylation status in the thymus and the inhibition of aortic Rcan1-4 mRNA expression (S7C and S7D Fig). These results strongly suggest that Ang-II activates molecular mechanisms critical for the early increase in BP that require Cn expression in SMCs, but not its phosphatase activity.

To determine whether the presence of Cn is required for long-term, sustained hypertension, we triggered Cn deletion in SMCs 7 days after inducing hypertension with Ang-II (Fig 6E). Treatment with Ang-II for 7 days elicited a similar degree of hypertension in Cn-Ctl and SM-Cn^f/f mice (Fig 6F). At this point, all mice were treated with Tx to elicit smooth muscle deletion of Cn in the SM-Cn^f/f mice, generating SM-Cn^−/− mice. BP decreased in SM-Cn^−/− mice as early as 1 day after the first Tx administration and continued to decline until the end of the experiment; in contrast, Cn-Ctl mice remained hypertensive until the end of the experiment (Fig 6F). These data strongly suggest that smooth muscle Cn is actively involved in the onset and maintenance of Ang-II-induced hypertension, identifying smooth muscle Cn as a potential target for the treatment of hypertension.

SMC Cn orchestrates the Ang-II-induced transcriptional program involved in arterial contractility and hypertension

To gain insight into the Cn-mediated molecular mechanisms underlying the early onset of hypertension, we performed a transcriptomic analysis to identify genes whose regulation by Ang-II in the aorta requires Cn expression in SMCs. To obtain an unbiased overview of gene expression changes in response to Ang-II, we selected all differentially expressed genes (DEGs) upon Ang-II treatment in each genotype. Specifically, we compared untreated versus Ang-II-treated Cn-Ctl mice and untreated versus Ang-II-treated SM-Cn^−/− mice, selecting protein-coding genes with a fold change (FC) > ±2. This set of genes was considered sensitive to Ang-II treatment. Heatmap analysis of these DEGs revealed that smooth muscle Cn is essential for the differential expression of most Ang-II-regulated genes in the aorta (Fig 7A). When analyzing DEGs independently of the FC threshold, we identified 800 DEGs in the aortas of Cn-Ctl mice treated for 2 h with Ang-II versus untreated mice; of these DEGs, almost 90% (722 genes) showed no evidence of Ang-II regulation in the aortas of SM-Cn^−/− mice (Figs 7A and S8A and S1 Table). To refine the list of candidate mediators involved in Ang-II-induced hypertension, we focused on a subset of 336 DEGs that were also differentially expressed between Ang-II-treated Cn-Ctl and SM-Cn^−/− mice (S8B Fig and S2 Table).

Since CsA pretreatment did not prevent Ang-II-induced hypertension, we reasoned that genes whose induction by Ang-II was sensitive to CsA would be unlikely to mediate hypertension. We therefore performed an additional transcriptomic analysis to identify genes whose regulation by Ang-II was unaffected by CsA. The performance of this study was very high and identified 2,653 DEGs in the aortas of mice treated with Ang-II for 2 h (S8C Fig and S3 Table). To obtain an ubiased overview of gene expression in this context, we selected protein-coding DEGs with an FC > ±2 from comparisons between untreated and Ang-II-treated WT mice, and between untreated and AngII-treated CsA-pretreated mice. Heatmap analysis of these DEGs showed a marked effect of Ang-II treatment on the aortic transcriptome and a subtle effect of CsA pretreatment on gene expression regulation by Ang-II (Fig 7B), with 2,596 of the Ang-II-regulated genes (almost 98%) insensitive to CsA pretreatment (S8C Fig). Comparison of these genes with the 336 Cn-dependent DEGs identified a group of 271 genes whose regulation by Ang-II required Cn expression but not its phosphatase activity (Fig 7C). Functional annotation clustering of these genes revealed enrichment in cluster terms strongly associated with cellular components involved in cell contractility regulation, such as “actin filament bundle”, “contractile fiber”, “focal adhesion”, and “myofibril” (Fig 7D).

VSMC contractility has a direct effect on BP regulation [3], and actomyosin cytoskeleton dynamics is critical for VSMC contractility regulation [24]. To investigate the influence of Cn on VSMC contractile capacity, we cultured primary Cn^f/f VSMCs transduced with GFP-encoding or Cre-encoding lentivirus (LV-GFP or LV-Cre, respectively) on collagen disks. In this assay, contractile capacity is determined by measuring the collagen disk surface area, which decreases as the attached cells contract in response to 3% fetal bovine serum (FBS) (Figs 7F and S9) [25,26]. Transduction with Cre-encoding LV efficiently deleted Cn expression (Fig 7E) and substantially impaired gel contraction relative to cells transduced with LV-GFP (Fig 7G and 7H). In contrast, transduction of WT VSMCs with an LxVP-encoding lentivirus (LV-LxVP) failed to prevent gel contraction (Fig 7G and 7H). These results suggest that Cn regulates VSMC contractility through a mechanism unrelated to its phosphatase activity. To investigate the contribution of SMC Cn to vessel contractility regulation in a more physiological setting, we measured the KCl-induced contraction of vehicle- or CsA-pretreated aortic and mesenteric resistance artery (MRA) rings from WT, Cn-Ctl, and SM-Cn^−/− mice. Cn deficiency, but not inhibition of its phosphatase activity with CsA, decreased aortic and MRA contractility in response to saturating (Fig 7I and 7J, left panels) or increasing KCl concentrations (Fig 7I and 7J, right panels), providing further support for the idea that Cn expression in SMCs is critical for arterial contractility regulation independently of Cn phosphatase activity.

To investigate the potential association of the group of 271 genes with hypertension, we performed an analysis using the Harmonizome dataset collection (http://amp.pharm.mssm.edu/Harmonizome). This analysis provided a hypertension score (HS) for >70% of these genes (S4 Table), suggesting a likely association with hypertension and supporting the validity of our transcriptomics approach as a way to identify genes encoding mediators of Ang-II-induced hypertension. Graphical representation of the FC in expression induced by Ang-II for the genes with the highest HS revealed a markedly different regulation in SM-Cn^−/− mice relative to Cn-Ctl, WT, and CsA-pretreated mice (Fig 8A). We further analyzed the expression of three genes with the highest HS—Serpine1, Ier3, and Gja1—in aortas from individual mice using RT-qPCR. After 2 h of Ang-II exposure, the expression of these genes was markedly inhibited in Cn-deficient mice but not in mice treated with CsA (S10 Fig), identifying them as candidate mediators of Ang-II-induced hypertension.

Consistent with the mRNA data, the expression of PAI-1, the protein encoded by Serpine1, was increased in aortas of mice treated with Ang-II for 2 h. This increase did not occur in SM-Cn^−/− mice and was not prevented by the treatment with CsA (Fig 8B and 8C). Multiple transcription factors can induce Serpine1 expression, including p53, AP1, NF-kB, CREB1, and SMAD2/3 [27–30]. Notably, SMAD2/3 activation occurs not only via TGF-β, but also through TGF-β-independent mechanisms triggered by Ang-II [31,32]. To explore the role of these factors in Cn-mediated induction of Serpine1 by AngII, we assessed Smad2 phosphorylation at Ser465/467, a marker of its activation. Smad2 phosphorylation was markedly increased after 2 h of Ang-II infusion in aortas from Cn-Ctl and CsA-treated mice but was not observed in aortas from SM-Cn^−/− mice (Figs 8D, 8E, S11A and S11B). These findings indicate that Smad2 activation by Ang-II is mediated by a phosphatase-independent activity of Cn.

To further evaluate the role of Smad2 in PAI-1 induction by Ang-II, we knocked down Smad2 in VSMCs. Smad2 silencing substantially reduced PAI-1 induction by Ang-II in VSMCs (Figs 8F, 8G and S11C), strongly suggesting that a structural role of Cn mediates Serpine1 induction through Smad2 activation.

PAI-1 is a key regulator of VSMC contractility and hypertension

Given the identification of PAI-1 as a major candidate mediator of Ang-II-induced hypertension, we investigated its potential role in regulating contractility and Ang-II-induced hypertension. Pre-treatment of VSMCs with the PAI-1 inhibitor TM5441 nearly abolished their capacity to contract collagen gels (Fig 8H and 8I), a result that was consistently replicated when Smad2 was silenced (Fig 8J and 8K).

In an in vivo approach, when mice were treated with PAI-1 inhibitor 24 h after the induction of hypertension with Ang-II (Fig 8L), there was a marked reduction in BP 24 h after the inhibitor administration (Fig 8M). Additionally, in a complementary experimental setup, acute intraperitoneal administration of TM5441 fully reversed hypertension induced by 7 days of Ang-II treatment (Fig 8N and 8O). These findings suggest that PAI-1 inhibition may rapidly restore normotension in hypertensive patients.

Taken together, our results emphasize the link between BP regulation and VSMC contractility, demonstrating that PAI-1 is a critical mediator of VSMC contractility and Ang-II-induced hypertension. Its expression is induced by Ang-II through Smad2 activation, which is dependent on a structural role of Cn in SMCs that does not require its phosphatase activity (Fig 8P).

Mouse strains

To delete Cn specifically in SMCs or ECs, we crossed Cnb1^flox/flox (Cn^f/f) mice [15] with either Cdh5-Cre^ERT2 transgenic mice [16] to obtain EC-Cn^f/f mice or with Myh11-Cre^ERT2 transgenic mice [17] to obtain SM-Cn^f/f mice. All mouse strains were backcrossed with C57BL/6J mice for more than eight generations to obtain a homogeneous genetic background and were maintained in homozygosity for the CnB1-floxed locus and in hemizygosity for the Cre^ERT2 transgene in the case of EC-Cn^f/f and SM-Cn^f/f mice. All mice were genotyped by PCR of genomic DNA obtained from tail samples (genotyping PCR). In some experiments, genomic DNA samples from Tx-treated mice were analyzed by agarose gel electrophoresis to detect the Cre-loxP recombination (recombination PCR). Primer sequences used for genotyping were: CnB1-floxed mice (5′-CCCTAGGCACTGTTCATGGT-3′, 5′-GCCACAGATTAGCCTCGTGT-3′, 5′-CACATTCACCCACAATTGC-3′), Myh11-Cre^ERT2 mice (5′-TGACCCCATCTCTTCACTCC-3′, 5′-AACTCCACGACCACCTCATC-3′, 5′-AGTCCCTCACATCCTCAGGTT-3′), and Cdh5-Cre^ERT2 mice (5′-GCCTGCATTACCGGTCGATGCAACGA-3′, 5′-GTGGCAGATGGCGCGGCAACACCATT-3′).

Animal procedures

Animal procedures were approved by the CNIC Ethics Committee and by the Madrid regional authorities (ref. PROEX 80/16 and PROEX 182.1/20) and conformed to European regulations regarding animal care (Directive EU 2010/63EU and Recommendation 2007/526/EC). All mice were housed in the CNIC specific pathogen-free animal facility with controlled temperature, humidity, and light-dark cycles. Sacrifice of mice was done by CO2 inhalation.

Cnb1^f/f-Cre^ERT2-neg together with Cnb1^wt/wt;Cdh5-Cre^ERT2 or Cnb1^wt/wt;Myh11-Cre^ERT2, were used as controls (Cn-Ctl) for Cnb1^f/f;Cdh5-Cre^ERT2 or for Cnb1^f/f;Myh11-Cre^ERT2, respectively. For inducible and conditional gene deletion, 10–12-week-old Cn-Ctl, SM-Cn^f/f, and EC-Cn^f/f male mice were given daily 1 mg i.p. injections of Tx (Sigma-Aldrich) on five consecutive days, generating SM-Cn^−/− and EC-Cn^−/− mice. Cn-Ctl, SM-Cn^−/−, and EC-Cn^−/− male mice were used after 6 weeks of Tx administration.

Ang-II (Sigma-Aldrich), CsA (Novartis), NE (Sigma-Aldrich), and TM5441 (MedChemExpress) were administered at 1 μg/kg/min, 5 mg/kg/d, 10 mg/kg/d, and 20 mg/kg/d, respectively, using subcutaneous osmotic minipumps (Alzet Corp, model 2001 or 2004). N_ω-nitro-l-arginine methylester hydrochloride (l-NAME; 0.5 g/l; Sigma-Aldrich, N5751) was administered in drinking water every 3 days. For minipump implantation, all mice were isoflurane-anesthetized (3%–5% isoflurane for induction, 1%–2% isoflurane for maintenance), and a small surgical pocket was made in the dorsal skin, where the minipump was implanted. Mice undergoing the same surgical procedure but without minipump implantation were used as controls for the Cn-Ctl, SM-Cn^−/−, EC-Cn^−/−, and CsA-treated groups.

For the AAA model, Cn-Ctl, SM-Cn^−/−, and EC-Cn^−/− male mice were fed a HFD (EF M HF coconut oil, + 7.5 g/kg Cholesterol Experimental diet, 38% chow, 10 mm; S9167-E011, SSNIFF) for 12 consecutive weeks. In the first week of feeding, Tx was injected to induce deletion in the Cre^ERT2-expressing mice. After 12 weeks of the HFD, this treatment was combined with the systemic infusion (1 µg/kg/min) of Ang-II (Sigma-Aldrich) with osmotic minipumps (Alzet Corp) [19].

For the SM-Cn^−/− transcriptome analysis, short-term Ang-II infusion and mouse selection were performed as follows: male mice were treated with Tx and, after 6 weeks, baseline BP measurements were performed. Osmotic minipumps with Ang-II (1 µg/kg/min) were implanted in Cn-Ctl and SM-Cn^−/− mice in parallel, and BP was measured after 2 and 24 h. Treated Cn-Ctl mice with systolic BP > 130 mmHg were considered valid for analysis and were euthanized; all the SM-Cn^−/− mice treated in parallel were selected for analysis, since none of them showed increased BP after Ang-II infusion. For the CsA transcriptome analysis, osmotic minipumps with CsA (CsA group) were implanted in WT C57BL/6J male mice. Minipumps were not implanted in control mice at this stage. After 5 days, baseline BP measurements were taken. Ang-II osmotic minipumps (1 µg/kg/min) were implanted in CsA and control mice in parallel, and BP was measured after 2 and 24 h. Ang-II-treated CsA and control mice with a systolic BP > 130 mmHg were considered valid for analysis and euthanized. If we could not confirm CnB1 deletion in any SM-Cn^−/− mouse or inhibition of NFAT dephosphorylation in the thymus of any CsA-treated mouse, those mice were removed from the experiment. Experiments for validation by RT-qPCR and protein expression by western blotting were performed in a similar way.

Blood pressure measurements

Arterial BP was measured by the mouse tail-cuff method using the automated BP-2000 Blood Pressure Analysis System (Visitech Systems, Apex, NC, USA). In brief, mice were trained for BP measurements every day for one week. After training, BP was measured one day before treatment to determine the baseline BP values in each mouse cohort. Measurements were repeated several times during experiments. BP measurements were recorded in mice located in a tail-cuff restrainer over a warmed surface (37 °C). Fifteen consecutive systolic and diastolic BP measurements were made, and the last 10 readings per mouse were recorded and averaged.

In vivo ultrasound imaging

Ultrasound images were obtained from isoflurane-anesthetized mice (1.5%–2% isoflurane) by high-frequency ultrasound with a VEVO 2100 echography device (VisualSonics, Toronto, Canada) at 30-micrometer resolution. Echocardiography was used to measure diastolic maximal internal aortic diameters and interventricular septum thickness in the parasternal long-axis view in the two-dimensional guided M-mode. Measurements were taken before treatment initiation to determine the baseline diameters and were repeated several times during the experiment. At endpoint, aortas were classified as AAA when they had increased in diameter by 50% or more [59,60], as dilated when the increase was 20%–50%, and as normal when the increase was 20% or less, relative to the initial measurement in all cases.

Lentivirus production and infection

LV expressing Cre recombinase, GFP protein, LxVP, and LxVPmut peptides have been described previously [7,18]. Pseudo-typed LV production and titration was as described previously [18,61]. Briefly, HEK-293T cells were transiently transfected using the calcium phosphate method, and culture supernatant was concentrated by ultracentrifugation (2 h at 26,000 rpm; Ultraclear Tubes; SW28 rotor and Optima L-100 XP Ultracentrifuge; Beckman). Viruses were suspended in cold sterile phosphate-buffered saline (PBS) and titrated by transduction of Jurkat cells for 48 h. Transduction efficiency (GFP-expressing cells) and cell death (propidium iodide staining) were quantified by flow cytometry (BD FACSCanto Flow Cytometer and FlowJo v10.8 software). The HEK-293T and Jurkat cell lines were purchased from ATCC. All cells were mycoplasma-negative.

Lentiviral transduction of aortas in vivo

WT male mice were anesthetized by intraperitoneal injection of ketamine (120 mg/kg) and xylazine (16 mg/kg), and a small incision was made to expose the jugular vein [7,61]. Virus solution (200 μl, 10⁹ particles/ml in PBS) was inoculated directly into the jugular vein 6 weeks before Ang-II minipump implantation. Transduction efficiency was analyzed postmortem in aortic samples by GFP immunohistochemistry.

Cell procedures

Primary mouse VSMCs were isolated and grown as described [62]. In brief, aortas from Cn^f/f male mice were dissected, and the adventitia was removed with forceps. Aortic tissue was cut into small rings and digested with 1 mg/ml collagenase type II (Worthington) and 0.5 mg/ml elastase (Worthington) in DMEM (Gibco) until single cells were observed under the microscope. The enzymatic reaction was stopped by the addition of DMEM supplemented with bovine serum. For primary mAECs, digested tissue was incubated with anti-ICAM-2 (BD Biosciences, 553326) conjugated to magnetic beads (Dynal Thermofisher, 11035). Isolated cells were cultured, and the remaining tissue was removed on the following days.

VSMCs were cultured in DMEM (Gibco) supplemented with 20% FBS (Gibco) and antibiotics. mAECs were cultured in DMEM F-12 (BioWhittaker BE, 12-719F) supplemented with 20% FBS, 100 mg/ml heparin (Sigma-Aldrich), 5 mg/ml EC growth factor (Gibco), and antibiotics on culture plates pre-covered with 0.5% gelatin and 100 mg/ml collagen type I (Sigma-Aldrich, C2124-50ML). EC purity was > 80% after 1 week of culture. All experiments were performed in passages 3–5, and all cells were mycoplasma-negative.

VSMCs were infected with specific LVs for 5 h at a multiplicity of infection = 10. The medium was then replaced with fresh supplemented DMEM. Seven days after the transduction, infection efficiency was evaluated and experiments were performed. In the case of Cre-recombinase-expressing LVs, infection was confirmed by immunoblot detection of Cn deletion. In the case of LxVP-expressing LVs, infection was confirmed by RT-qPCR detection of GFP.

VSMCs were transfected with siRNA using Lipofectamine Transfection Reagent (Invitrogen, 13778075) following the manufacturer’s protocol. Smad2-specific siRNAs (ON-TARGETplus Mouse Smad2 siRNA (SMARTPool) L-040707-00-0005, Dharmacom) targeted the following sequences: 5′-ACUCAGAAUUGCAAUACUA-3′, 5′-CGAAUGUGCACCAUAAGAA-3′, 5′-GCUCAAGCAUGUCGUAAAC-3′, and 5′-GAAAGGGUUGCCACAUGUU-3′. Non-targeting control siRNAs (ON-TARGETplus Non-targeting Pool, D-001810-10-05, Dharmacom) targeted the sequences: 5′-UGGUUUACAUGUCGACUAA-3′, 5′-UGGUUUACAUGUUGUGUGA-3′, 5′-UGGUUUACAUGUUUUCUGA-3′, and 5′-UGGUUUACAUGUUUUCCUA-3′. Silencing efficiency was confirmed via immunoblot analysis, where Smad2 protein levels were evaluated in lysates from VSMCs transfected with Smad2 siRNAs. The results demonstrated effective knockdown of Smad2 expression, validating the transfection protocol.

For collagen contraction assays, 150 µl aliquots of PureColTM EZ Gel Solution (Sigma-Aldrich, 5074) were polymerized for 1 h at 37 °C in p48 wells, and 8 × 10⁴ VSMCs were seeded on each gel. Cells were left to adhere to the gels for 5 h and then starved for 72 h. The gel–cell composites were then gently detached from the plates and incubated in DMEM supplemented with 3% FBS for 24—48 h. When indicated, cells were pretreated with 50 µM TM5441 (MedChemExpress) for 2 h. At the end of the experiment, gels were washed in PBS and fixed with 3% paraformaldehyde (PFA) for 15 min. Gel images were taken with a Nikon SMZ800 scoop camera (0.8× objective). Each condition was replicated in 3–4 wells, and each condition was replicated with different batches of VSMCs. Gel contraction was quantified with ImageJ software as follows: each gel area was quantified and normalized to the average of all the areas in a single experiment; the means of the normalized areas for each condition are presented.

Myography

Rings (3 mm long) from control (Cn-Ctl or WT) and SM-Cn^−/− male mouse thoracic descending aortas and superior MRA were excised and placed on a wire myograph (model 610M, Danish Myo Technology, Aarhus, Denmark) for the measurement of isometric tension. The organ chamber was filled with Krebs solution (composition in mM: NaCl 118, KCl 4.75, NaHCO₃ 25, MgSO₄ 1.2, CaCl₂ 2, KH₂PO₄ 1.2, and glucose 11) and infused with carbogen (5% CO₂ and 95% O₂, pH ∼7.4), at 37 °C. When indicated, CsA (200 ng/ml) was added to WT arteries at the time of removal from the animal and was maintained during ring excision and throughout the experiment in the chamber. Aortic and MRA rings were stretched to tensions of 5 and 3 mN, respectively, and equilibrated for 30 min. Tension characteristics were obtained with the myograph software (LabChart). Aortic and MRA rings were initially stimulated with 80 and 50 mM KCl, respectively, followed by determination of KCl concentration-response curves (1–100 mM). Vasoconstriction measurements are presented as the force (mN) exerted by the rings.

Aortic histology

After sacrifice of mice by CO₂ inhalation, aortas were perfused with saline, isolated, and fixed in 4% PFA overnight at 4 °C. Paraffin cross sections (5 μm) from fixed aortas were stained with Masson’s trichrome (Masson), Hematoxylin and Eosin (HE), or Elastic Verhoeff–Van Gieson (EVG) stain. Medial wall thickness was determined from HE staining by quantifying the area of the media in four non-consecutive sections containing complete aortic cross-sections using the NDP.view2 Image viewing software.

For immunohistochemistry, deparaffinized sections (5 µm) were rehydrated, boiled to retrieve antigens (10 mM citrate buffer, pH 6), and blocked for 1 h with 5% goat serum plus 2% BSA in PBS. Samples were incubated with anti-GFP primary antibody (Invitrogen, A11122, 1:200) overnight at 4° and then with a biotinylated secondary antibody for 1 h at room temperature. Signal was detected with the DAB (3,3′-Diaminobenzidine) reaction (Vector Laboratories) for a maximum 7-min reaction time. Sections were then counterstained with hematoxylin, dehydrated, and mounted with DPX (CasaÁlvarez). For immunofluorescence, deparaffinized sections (5 µm) were rehydrated, boiled for antigen retrieval (10 mM citrate buffer, pH 6), and blocked for 1 h with 5% goat serum plus 2% BSA plus 5% horse serum in PBS-Triton 0.01% for anti-Ki67, anti-CD68, and anti- α-SMA. For p-Smad2, DNA was denatured with 1.5M HCl for 20 min RT, followed by blocking with 10% goat serum plus 2% BSA plus 10% horse serum in PBS-Triton 0.01%. Samples were incubated overnight at 4 °C with with the following primary antibodies: anti-Ki67 (ab15580, Abcam), anti-CD68 (ab15580, Abcam), and anti-phospho-Smad2 (Ser465/467; AB3849-I, Millipore) primary antibodies. Bound primary antibodies were detected with Alexa Fluor 594-conjugated or 647-conjugated secondary antibodies (Thermo Fisher, 1:500). Cy3 conjugated anti-α-SMA (C6198, Sigma, 1:2000) was incubated for 1 h RT. DAPI (1 ng/ml; Invitrogen) was used to label cell nuclei. Images were captured using a confocal microscope (Leica, SP5). For quantification, the medial layer of the aorta was defined as the region of interest (ROI) based on the elastin signal. ImageJ software was used to measure the mean intensity of α-SMA staining, and an ImageJ macro quantified nuclear p-Smad2 intensity. CD68-positive cells and Ki67-positive nuclei were manually counted and averaged per section. Data represent the average of at least two sections per mouse.

In situ southwestern histochemistry was performed as previously described [7]. Briefly, 5 μm aortic cross sections were deparaffinized and rehydrated. Sections were fixed with 0.5% PFA for 25 min at 28 °C, and endogenous alkaline phosphatase was quenched with 5 mM levamisole (L9756, Sigma-Aldrich). Preparations were then digested with 0.5% pepsin A (P7000, Sigma-Aldrich) in 1 N HCl for 35 min at 37 °C and washed twice with HEPES/BSA buffer (10 mM HEPES, 40 mM NaCl, 10 mM MgCl₂, 1 mM dithiothreitol, 1 mM EDTA, and 0.25% BSA, pH 7.4). Sections were incubated with 0.1 mg/ml DNase I in HEPES/BSA buffer for 30 min at 30 °C. Activated NFAT proteins were probed with the synthetic sense DNA sequence 5′-GATCGCCCAAAGAGGAAAATTTGTTTCATACAG-3′. This sequence contains a composite site comprising the NFAT and AP1 sites from the mouse IL-2 promoter [63]. The NFAT probe was labeled with digoxigenin (DIG) (Sigma-Aldrich) and diluted to 25 pM in HEPES/BSA buffer containing 0.5 mg/ml poly (dI-dC) (P4929, Sigma-Aldrich). Absence of probe was used as negative control. After overnight incubation at 37 °C in a humidified chamber, sections were washed twice with buffer 1 (0.1 M maleic acid, 0.15 M NaCl, 0.03% Tween-20, pH 7.5) and incubated for 1 h in blocking solution (0.1% SSC and 0.1% SDS diluted 1:10 in buffer 1 without Tween). The preparations were washed with buffer 1 and incubated for 2 h at 37 °C with an anti-DIG antibody conjugated to alkaline phosphatase (1:200 in blocking solution; 11093274910, Roche). Next, sections were washed in buffer 1 and buffer 3 (Tris HCl 0.1 M, 0.1 M NaCl, and 50 mM MgCl₂, pH 9.5) at room temperature. Bound alkaline phosphatase was visualized with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP; 11681451001, Roche). The reaction was stopped by incubation in buffer 4 (10 mM Tris and 1 mM EDTA, pH 8.0), and sections were dehydrated through a graded ethanol series and mounted in 50% glycerol in PBS.

All bright-field images were obtained with a Leica DM2500 microscope with 20× and 40× HCX PL Fluotar objectives and with Leica Application Suite V3.5.0 software. Southwestern histochemistry images were analyzed and quantified with ImageJ software as follows: after color deconvolution by fast red blue DAB, the blue channel was selected (color 2). The same threshold was selected in all images, and only the medial aortic region was selected as a ROI and measured. Raw data are presented. Layouts were obtained in Adobe Illustrator.

Immunoblotting

Mouse samples were obtained by dissection, and in the case of aortas, perivascular tissue and adventitia were removed with forceps. The tissue was frozen in liquid nitrogen and then homogenized using a mortar. Cultured cells were washed with ice-cold PBS and frozen at −80 °C. Protein extracts from tissue samples and cells were obtained in ice-cold high-salt lysis buffer (20 mM Tris HCl pH 7.5, 5 mM MgCl₂, 50 mM NaF, 10 mM EDTA, 0.5 M NaCl, 1% Triton X-100) for the detection of CnB, CnA, tubulin, α-SMA, Cd31, and NFATc3, and in RIPA buffer (150 mM NaCl, 10mM Tris HCl pH8, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate and 5 mM EDTA) for the detection of PAI-1, phospho-Smad2 (Ser465/467), Smad2/3 and tubulin. Both lysis buffers were supplemented with protease, phosphatase, and kinase inhibitors. Homogenates were centrifuged for 15 min at 16,000g at 4 °C, and proteins were collected in the supernatants. Protein was quantified with the Lowry assay kit. Protein samples (30 μg) were separated under reducing conditions on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Proteins were detected with the following primary antibodies: mouse monoclonal anti-CnB (Sigma, 0581, 1:1000), rabbit polyclonal anti-CnA (Merck Millipore, 07-1491, 1:2000), mouse monoclonal anti-tubulin (Sigma, T6074, 1:40000), rabbit polyclonal anti-Cd31 (Abcam, ab28364, 1:1000), mouse monoclonal anti α-SMA (Sigma, A5228; 1:5000), rabbit polyclonal anti-NFATc3 (M-75) (SCB, sc-8321, 1:1000), rabbit monoclonal anti-PAI-1 (Abcam, ab222754, 1:500), anti-phospho-Smad2 (Cell Signaling 3108, 1:1000), and anti-Smad2/3 (Cell Signaling, 8685, 1:1000). After washing and incubation with the appropriate specific HRP-conjugated secondary antibodies, immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) detection reagent (GE Healthcare, Chicago, IL, USA, RPN2106) or acquired in the ImageQuant LAS 4000 device using the Immobilon Forte Western HRP substrate (WBLUF0100, Millipore). Quantification of protein band intensity was performed with ImageJ using Tubulin band intensity for normalization.

RT and quantitative PCR

Aortas were extracted after perfusion with 5 ml saline solution, and the adventitia layer was discarded. Frozen tissue was homogenized using a mortar and an automatic bead homogenizer (MagNA Lyzer). Total RNA was isolated with TRIZOL (Life Technologies). Total RNA (1 μg) was reverse transcribed at 37 °C for 50 min in a 20 µl reaction mix containing 200U Moloney murine leukemia virus (MMLV) reverse transcriptase (Life Technologies), 100 ng random primers, and 40U RNase Inhibitor (Life Technologies). The resulting cDNA was analyzed by quantitative real-time PCR (qPCR) in an AB7900 sequence detection PCR system (Applied Biosystem, Foster City, CA, USA) using SYBR Green PCR or TaqMan Universal PCR master mixes (Go Taq Probe PCR Master Mix; Promega, A600A or A610A, respectively). The following TaqMan probes were used: Ptgs2 (Mm00478374_m1) and Hprt1 (Mm00446968_m1). The specific probes used for SYBR Green detection were as follows: Gja1 (5′-CTTGGGGTGATGAACAGT-3′; 5′-TGAGCCAAGTACAGGAGT-3′ [64]), Hprt (5′-GCTGGTGAAAAGGACCTCT-3′; 5′-CACAGGACTAGAACACCTGC-3′), Ier3 (5′-GCGCGTTTGAACACTTCTCG-3′; 5′-TGGCGCCGGACCACTC-3′), Rcan1–4 (5′-TTGTGTGGCAAACGATGATGT-3′; 5′-CCCAGGAACTCGGTCTTGT-3′), Serpine1 (5′-GAGGTAAACGAGAGCGGCA-3′; 5′-AAGAGGATTGTCTCTGTCGGG-3′). Probe specificity was checked by post-amplification melting-curve analysis in the case of SYBR amplification; for each reaction, only one Tm peak was produced. The amount of target mRNA in samples was estimated by the 2^−∆CT relative quantification method, using Hprt for normalization, and represented in graphs. Each sample represented belongs to one mouse aorta.

RNA sequencing

Aortas were isolated after perfusion with saline, and the perivascular tissue and the adventitia layer were removed with forceps. A piece of aortic tissue was used for PCR confirmation of CnB1 recombination, and the thymus was isolated for protein extraction to check the NFAT phosphorylation state, when appropriate. Aortic tissue was frozen in liquid nitrogen and homogenized with lysis buffer (Qiagen, 64204) using an automatic bead homogenizer (MagNA Lyser; Roche). Total RNA was isolated with the RNeasy MinElute Cleanup Kit (QIAGEN, 74204). Each triplicate was a pool of three aortas (total, nine aortas per condition). RNA quality and purity were analyzed by Nanodrop and Bioanalyzer (Agilent 2100 Bioanalyzer RNA NanoChip). Only RNA samples with appropriate absorbance ratios were used (260/280 nm > 2.0; 230/260 nm 2.0–2.2); RNA samples with a RIN (RNA Integrity number-Bioanalyzer) below seven were discarded. For each condition, 1.5 μg of RNA was retro-transcribed with oligo dT priming, and the cDNA obtained was used to generate the transcriptome library. The library was sequenced using Illumina technology (Illumina Genome Analyzer IIx System). Library generation and sequencing were performed by the CNIC Genomics Unit under the supervision of Dr. Ana Dopazo. Reads were aligned first using BoWTie version 0.12.7 and then mapped to the latest Ensembl transcript set using RSEM v1.16. One replicate of the untreated Cn-Ctl group showed a high deviation from the other two replicates in PCA analysis and was therefore discarded. Genes showing altered expression with an adjusted p < 0.05 were considered DEGs. Only protein-coding genes were included in further analysis. Gene expression data are available at http://www.ncbi.nlm.nih.gov/projects/geo/ under accession number GSE255061.

Venn diagrams were generated with the online software Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html), and the DEGs represented in each diagram are indicated in the figure and figure legend. Heatmaps were generated using R library ‘stats’ [65] and ‘gplots’ [66] to represent mean normalized mRNA expression of the DEGs regulated by Ang-II in Cn-Ctl, SM-Cn^−/−, WT, and CsA-pretreated mice versus the untreated condition (FC > ±2). Mean normalized mRNA expression was obtained from triplicates of each condition. The HS was extracted automatically by an exact pattern match using the gen symbol as the identifier. The processed database was the Harmonizome dataset collection [67] for the curated CTD Gene-Disease Associations related to the term ‘Hypertension’, where the standardized value was renamed the HS.

Functional enrichment analysis

Gene Ontology (GO) enrichment analysis of DEGs was performed using g:Profiler software [68]. The analysis mainly focused on gen function data sources related to cellular components (GO:CC). We were able to map genes to statistically significant enriched terms. The statistical domain scope was only applied to annotated genes, adjusting the significance threshold by multiple testing (using the g:Profiler tailor-made algorithm g:SCS), set to a value p < 0.05. This approach has produced more accurate results than obtained with the standard Bonferroni correction or BH-False Discovery Rate methodologies [69]. To select the most relevant functional categories, the term size was adjusted to 0 as minimum and 300 as maximum, avoiding redundant and generic terms.

Statistical analysis

GraphPad Prism software (versions 9.4.1 and 10.1.2) was used for the statistical analysis. Appropriate tests were chosen according to the data distribution based on the Saphiro–Wilk normality test. Outliers were identified and excluded by the ROUT method.

Comparisons for measured parameters and repeated measurements (RM) are indicated in each figure legend. Differences were analyzed by unpaired Student t test, Mann-Whitney test, and one-way ANOVA or two-way ANOVA tests, and multiple comparisons were analyzed with the Tukey or Šídák post hoc tests, as appropriate; the tests used are indicated in each figure legend. Differences were considered statistically significant at p < 0.05.

The number of animals used is indicated in the corresponding figures or figure legends. No statistical method was used to predetermine sample size. Sample size was chosen empirically according to our experience in the calculation of experimental variability. All experiments were carried out with at least three biological replicates.

Experimental groups were balanced in terms of animal age and weight. Only male mice were used because the Myh11-Cre^ERT2 transgene was inserted in the Y chromosome (https://www.jax.org/strain/019079). Investigators were not blinded to group allocation during experiments or to outcome assessments. Animals were genotyped before experiments, caged together, and treated in the same way.

S1 Fig. Ang-II administration in mice does not modify Cn expression in the aorta.

(A) Representative immunoblot analysis of CnA, CnB and tubulin (loading control) and (B) quantification of their relative expression in protein extracts from untreated Cn-Ctl (n = 3–4) and Ang-II-treated Cn-Ctl (n = 4–7), Molecular weights (kDa) are indicated. Each data point denotes an individual mouse, and data in histograms are presented as mean ± s.e.m. unpaired Student t test. Underlying data can be found in S1 Data.

https://doi.org/10.1371/journal.pbio.3003163.s001

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S2 Fig. Cn deletion in SMCs does not prevent HFD-induced body weight gain.

Body weight of 29 Cn-Ctl, 19 EC-Cn^−/−, and 28 SM-Cn^−/− mice fed a chow diet and 46 Cn-Ctl, 15 EC-Cn^−/−, and 18 SM-Cn^−/− mice after 12 weeks of HFD. Each data point denotes an individual mouse, and the horizontal bars denote the mean (long bar) and the s.e.m. ****p < 0.0001, ***p < 0.001; one-way ANOVA with Tukey’s multiple comparison post hoc test. Underlying data can be found in S1 Data.

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S3 Fig. Immunofluorescence characterization of the AbAo in the AAA mouse model.

(A) Representative images of α-SMA immunostaining in AbAo cross-se ctions from the indicated groups of mice, and (B) quantification of α-SMA mean intensity in the same cross-sections. Representative images of (C) Ki67 (arrowheads), and (D) CD68 (arrowheads) immunostaining in AbAo cross sections from the indicated groups of mice, with quantification of (E) Ki67 + nuclei and (F) CD68+  cells in the same cross sections. (A,C,D) L, lumen. Scale bar, 100 μm. (B,E,F) Each data point denotes an individual mouse, with values representing the average quantification of at least 2 independent images per mouse. Data in histograms are presented as mean ± s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05; two-way ANOVA with Šídák’s post hoc test (B); Mann–Whitney and unpaired Student t test (E, F). Underlying data can be found in S1 Data.

https://doi.org/10.1371/journal.pbio.3003163.s003

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S4 Fig. Cn deletion in SMCs inhibits AngII-induced diastolic hypertension.

Diastolic BP values at the indicated times in 5 Cn-Ctl, 3 EC-Cn^−/−, and 5 SM-Cn^−/− control-treated mice and 22 Cn-Ctl, 10 EC-Cn^−/−, and 18 SM-Cn^−/− Ang-II-treated mice. Data are mean ± s.e.m. ****p < 0.0001, ***p < 0.001, **p < 0.01 vs. SM-Cn^−/− Ang-II, ^###p < 0.001 vs. Cn-Ctl control, ^##p < 0.01, ^#p < 0.05 vs SM-Cn^−/− or Cn-Ctl control; RM two-way ANOVA with Tukey’s post hoc test. Underlying data can be found in S1 Data. Data from untreated and Ang-II-treated Cn-Ctl mice were repeated in left and right panels as indicated in S1 Data.

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S5 Fig. Cn deletion in SMCs inhibits norepinephrine (NE)- and L-NAME-induced AHT.

(A) Experimental design: 10–12-week-old mice were treated with tamoxifen for 5 consecutive days (open arrow heads) and, after 6 weeks, NE osmotic minipumps were implanted for 4 weeks in one group of mice; control mice were operated without minipump implantation. Another mouse group was treated with L-NAME in drinking water for 4 weeks. BP was measured at the indicated time points (purple arrows), and mice were euthanized at the end of the experiment. (B) Systolic BP values of 10 Cn-Ctl and 5 SM-Cn^−/− control-treated mice, 11 Cn-Ctl and 6 SM-Cn^−/− NE-treated mice, and 5 Cn-Ctl and 6 SM-Cn^−/− L-NAME-treated mice. Data are means ± s.e.m. ***p < 0.001, **p < 0.01, *p < 0.05 vs. treated SM-Cn^−/−, ^####p < 0.001 vs. control Cn-Ctl and ^#p < 0.05 vs. control SM-Cn^−/−; RM two-way ANOVA with Tukey’s post hoc test. Underlying data can be found in S1 Data. Data from untreated Cn-Ctl and untreated SM-Cn−/− mice are repeated in top and bottom panels as indicated in S1 Data.

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S6 Fig. Cn inhibition by CsA impairs Ang-II-induced NFAT dephosphorylation and cardiac hypertrophy.

(A) Representative NFATc3 immunoblot analysis of thymus extracts from WT mice treated as indicated (top panel) and Ponceau staining of the membrane (bottom panel). Hyper-phosphorylated NFATc3 (P-NFAT) and de-phosphorylated NFATc3 (NFAT) are indicated. (B) End-of-experiment heart weight vs. body weight ratio (HW/BW) values. Each data point denotes an individual mouse, and data in histograms are presented as mean ± s.e.m. ****p < 0.0001, ***p < 0.001; two-way ANOVA with Šídák’s post hoc test. (C) Echocardiography-determined interventricular septum (IVS) thickness before CsA (day −2) and Ang-II (day 0) administration and after 21 days of Ang-II treatment. Each data point denotes an individual mouse, and the horizontal bars denote the mean (long bar) and the s.e.m. ****p < 0.0001, **p < 0.01; RM two-way ANOVA with Tukey’s post hoc test. (B, C) Ang-II (n = 10), CsA + Ang-II (n = 8), control (n = 8), and CsA (n = 5). Underlying data can be found in S1 Data.

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S7 Fig. Cn deletion, but not inhibition of Cn phosphatase activity, prevents Ang-II-induced diastolic hypertension.

(A, B) Diastolic BP was measured at the indicated time points in the same Cn-Ctl, SM-Cn^−/−, and non-pretreated and CsA-pretreated WT mice as in Fig 6B (A) and Fig 6D (B). Each data point denotes the BP value in an individual mouse, and the horizontal bars denote the mean (long bar) and the s.e.m. (n = 9 mice per group and per time point). ****p < 0.0001, **p < 0.01, *p < 0.05 vs. baseline; RM two-way ANOVA with Tukey’s post hoc test. ^##p < 0.01 vs. 2 or 24 h Ang-II Cn-Ctl; RM two-way ANOVA with Šídák’s post hoc test. (C) Representative NFATc3 immunoblot of thymus extracts from mice treated as indicated. Hyper-phosphorylated NFATc3 (P-NFAT) and de-phosphorylated NFATc3 (NFAT) are indicated. Ponceau staining of the membrane is shown below. (D) Quantification of mRNA expression, as assessed by RT-qPCR, in extracts from the aorta of untreated control (3 Cn-Ctl plus 3 WT), SM-Cn^−/− (n = 5), and CsA-pretreated (n = 6) mice and Ang-II-treated control (3 Cn-Ctl plus 3 WT), SM-Cn^−/− (n = 5), and CsA-pretreated (n = 6) mice. Each data point denotes an individual mouse, and data in histograms are presented as mean ± s.e.m. **p < 0.01, ****p < 0.0001; two-way ANOVA with Šídák’s post hoc test. Underlying data can be found in S1 Data.

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S8 Fig. Selection of potentially AHT-related genes from the transcriptomic analyses.

(A) 800 DEGs in Cn-Ctl mice treated with Ang-II for 2 h were selected as potential mediators of AHT, and 130 DEGs regulated in SM-Cn^−/− mice treated with Ang-II for 2 h were excluded, leaving 722 potentially AHT-related genes, as indicated in the Venn diagram (below). (B) Of these genes, only those whose expression differed between the hypertensive condition (Cn-Ctl Ang-II 2 h) and the non-hypertensive condition (SM-Cn^−/− Ang-II 2 h) were selected using a Venn diagram (below). The figure indicates the resulting 336 DEGs potentially involved in AHT and whose regulation is Cn-expression dependent. (C) 2,653 DEGs regulated in WT mice by treatment with Ang-II for 2 h were selected as potential mediators of AHT. Only those whose expression was similar in both hypertensive conditions (not showing differential expression between WT Ang-II 2 h and CsA Ang-II 2 h) and CsA-independent were selected using a Venn diagram (below). The figure indicates the resulting 2,596 DEGs potentially involved in AHT and whose regulation is independent of Cn phosphatase activity.

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S9 Fig. Collagen contraction assay.

(A) Representative images from three independent experiments and (B) quantification of the surface area of fixed collagen gels in the absence or presence of cells stimulated as indicated. Each data point denotes the mean of each experiment, and data in histograms are presented as mean ± s.e.m. *p < 0.05 by two-way ANOVA with Šídák’s post hoc test. No cells without serum (n = 2), starved cells (n = 3), No cells with 3% FBS (n = 3), Cells with 3% FBS (n = 3). Underlying data can be found in S1 Data.

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S10 Fig. Serpine1, Gja1, and Ier3 induction is mediated by a structural role of SMC Cn.

RT-qPCR analysis of mRNA expression in aortic extracts from untreated control (3 Cn-Ctl plus 3 WT), SM-Cn^−/− (n = 5), and CsA-pretreated (n = 6) mice and from Ang-II-treated control (3 Cn-Ctl plus 3 WT), SM-Cn^−/− (n = 5), and CsA-pretreated (n = 6) mice. Two-way ANOVA with Šídák post hoc test; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Underlying data can be found in S1 Data.

https://doi.org/10.1371/journal.pbio.3003163.s010

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S11 Fig. Smad2 activation is mediated by a structural role of SMC Cn.

(A) Representative immunoblot analysis of phospho-Smad2, Smad2/3 and tubulin (loading control) and (B) quantification of their relative expression in protein extracts from untreated control (n = 3) and SM-Cn^−/− (n = 3) mice; CsA-pretreated (n = 3) mice; and Ang-II-treated control (n = 4), SM-Cn^−/− (n = 4), and CsA-pretreated (n = 4) mice. Molecular weights (kDa) are indicated. Each data point denotes an individual mouse, and data in histograms are presented as mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; two-way ANOVA with Šídák post hoc test. (C) Quantification of the relative expression in protein extracts from the representative immunoblot in Fig 8F (n = 3 independent experiments) with each data point representing an individual replicate. Data are presented as mean ± s.e.m. ***p < 0.001, unpaired Student t test. Underlying data can be found in S1 Data.

https://doi.org/10.1371/journal.pbio.3003163.s011

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