Vascular Smooth Muscle Cell-Specific Progerin Expression Provokes Contractile Impairment in a Mouse Model of Hutchinson-Gilford Progeria Syndrome that Is Ameliorated by Nitrite Treatment

Cardiovascular disease (CVD) is the main cause of death worldwide, and aging is its leading risk factor. Aging is much accelerated in Hutchinson–Gilford progeria syndrome (HGPS), an ultra-rare genetic disorder provoked by the ubiquitous expression of a mutant protein called progerin. HGPS patients die in their teens, primarily due to cardiovascular complications. The primary causes of age-associated CVD are endothelial dysfunction and dysregulated vascular tone; however, their contribution to progerin-induced CVD remains poorly characterized. In the present study, we found that progeroid LmnaG609G/G609G mice with ubiquitous progerin expression show both endothelial dysfunction and severe contractile impairment. To assess the relative contribution of specific vascular cell types to these anomalies, we examined LmnaLCS/LCSTie2Cretg/+ and LmnaLCS/LCSSm22αCretg/+ mice, which express progerin specifically in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. Whereas vessel contraction was impaired in mice with VSMC-specific progerin expression, we observed no endothelial dysfunction in mice with progerin expression restricted to VSMCs or ECs. Vascular tone regulation in progeroid mice was ameliorated by dietary sodium nitrite supplementation. Our results identify VSMCs as the main cell type causing contractile impairment in a mouse model of HGPS that is ameliorated by nitrite treatment.


Introduction
Cardiovascular disease (CVD) is the leading cause of death worldwide, in part due to progressive aging, the main CVD risk factor [1]. A number of additional factors have been identified that increase the risk of developing CVD, either acting alone or in combination (e.g., hypercholesterolemia, diabetes, sedentary lifestyle, and smoking) [2][3][4]. However, studies on the effects of age alone (the only factor we cannot modify or treat) remain scarce due to their high research costs associated with the necessary long-term resource investment and delays in collecting results. The study of premature aging syndromes characterized by accelerated CVD thus offers a unique opportunity to investigate age-dependent drivers of CVD in the absence of other confounding risk factors [5,6]. ethics committees and the Animal Protection Area of the Comunidad Autónoma de Madrid (PROEX 135/14). Mice were housed at the CNIC pathogen free facility and sacrificed at 14-15 weeks of age.

Nitrite Treatment
When indicated, 6-week-old Lmna G609G/G609G mice were treated for 8 weeks with sodium nitrite (NaNO 2 , Sigma-Aldrich, St. Louis, MO, USA). The compound was dissolved in drinking water at a final concentration of 50 mg/L, a dose that has been reported to be safe in mice, showing no evidence of toxicological or carcinogenic effects and no effect on water consumption [38]. Consistent with these findings, we observed no adverse effects or changes in water consumption in Lmna G609G/G609G or Lmna +/+ mice treated with sodium nitrite.

Wire Myography
Animals were euthanized at 14-15 weeks of age by CO 2 inhalation. Immediately after sacrifice, the thoracic and abdominal cavities were opened. Thoracic aortas were excised and immediately placed in ice-cold Krebs-Henseleit solution (KHS: 115 mM NaCl, 2.5 mM CaCl 2 , 4.6 mM KCl, 1.2 mM KH 2 PO4, 1.2 mM MgSO 4 , 25 mM NaHCO 3 , 11.1 mM glucose, and 0.01 mM EDTA), gently cleaned of fat and connective tissue, and cut into~2 mm long segments. Aortic rings were then mounted on 2 tungsten wires in a wire myograph system (620M, Danish Myo Technology A/S, Hinnerup, Denmark) and immersed in 37 • C KHS with constant gassing (95% O 2 and 5% CO 2 ). Wire myography was performed as previously described [39]. Optimal vessel distension was determined by normalization using the Laplace Equation (Tension = [pressure × radius]/thickness) to calculate the position at which the tension was equivalent to an intraluminal pressure of 100 mmHg (L100) [39]; vessels were then set up to the optimal tension (physiological distension, 0.9 of L100).
After equilibration for 30 min, vasoconstriction was studied by exposing the aortic rings first to 120 mM KCl and then to increasing doses of phenylephrine (from 1 nm to 10 µM; Sigma-Aldrich). We assessed the contribution of vessel stiffness to the contractile function by analyzing contraction induced by 120 mM KCl before and after collagen degradation with collagenase type II (0.2% w/v, 15 min incubation; Thermo Fisher Scientific, Waltham, MA, USA). Endothelium-dependent vasodilation was assessed by examining the response to increasing doses of acetylcholine (from 0.1 nM to 10 µM; Sigma-Aldrich) in segments previously contracted with 1 µM phenylephrine. Endothelium-independent vasodilation induced by increasing doses of the NO donor diethylamine NONOate (DEA-NO) (from 0.1 nM to 10 µM; Sigma-Aldrich) was examined in segments previously contracted with 1 µM phenylephrine. Drug treatments were separated by extensive washes and a stabilization period of at least 15 min. When indicated, the contribution of NO, Prostacilin I 2 (PGI 2 The results of wire myography experiments are represented as dose-response curves and as area  under the curve (AUC), which provides information regarding differences in the dose-response curve as a whole and in a continuous manner.

Statistical Analysis
Results are represented as mean ± standard error of the media (SEM), and statistical differences were analyzed using the tests indicated in the figure legends.

Impaired Vascular Function in Progeroid Mice with Ubiquitous Progerin Expression
To investigate the impact of ubiquitous progerin expression on vascular function, we performed ex vivo wire myography experiments to examine contraction and dilation in thoracic aorta segments obtained from 14-15-week-old Lmna G609G/G609G mice and age-matched wild-type (Lmna +/+ ) littermate controls. These studies demonstrated impaired contraction of progeroid vessel segments in response to incubation with phenylephrine ( Figure 1A) and KCl ( Figure 1B).
Cells 2020, 9, x 4 of 12 stabilization period of at least 15 min. When indicated, the contribution of NO, Prostacilin I2 (PGI2), H2O2, and O2 − to endothelium-dependent vasodilation was assessed by adding the following agents to the bath 30 min before the acetylcholine dose-response curve: 0.1 mM L-NAME (NOS inhibitor), 10 µM tranylcypromine (PGI2 inhibitor), 2000 U/mL catalase (H2O2 decomposing agent), and 0.1 mM Tempol (O2 − scavenger) (all from Sigma-Aldrich). All drugs were dissolved in water except for the Tempol stock solution, which was prepared in ethanol.
The results of wire myography experiments are represented as dose-response curves and as area under the curve (AUC), which provides information regarding differences in the dose-response curve as a whole and in a continuous manner.

Statistical Analysis
Results are represented as mean ± standard error of the media (SEM), and statistical differences were analyzed using the tests indicated in the figure legends.

Impaired Vascular Function in Progeroid Mice with Ubiquitous Progerin Expression
To investigate the impact of ubiquitous progerin expression on vascular function, we performed ex vivo wire myography experiments to examine contraction and dilation in thoracic aorta segments obtained from 14-15-week-old Lmna G609G/G609G mice and age-matched wild-type (Lmna +/+ ) littermate controls. These studies demonstrated impaired contraction of progeroid vessel segments in response to incubation with phenylephrine ( Figure 1A) and KCl ( Figure 1B).  Strength of contraction induced by KCl. Statistical differences were analyzed by two-way ANOVA with Bonferroni's post-hoc test for phenylephrine data, and by two-tailed t-test for KCl data. * p < 0.05 *** p < 0.001.
To assess endothelial function, we first exposed phenylephrine-precontrated aortas to the endothelium-dependent vasodilator acetylcholine ( Figure 2A). Relaxation induced by the physiological acetylcholine dose (0.1 µM) was significantly lower in Lmna G609G/G609G vessel segments, a result also evidenced by a difference in logEC50 (−7.025 ± 0.06 in Lmna +/+ aortic rings versus −6.511 ± 0.09 in Lmna G609G/G609G aortic rings) (Figure 2A, left) and a lower AUC for acetylcholine-induced relaxation (Figure 2A, right). In contrast, there were no significant differences in the relaxation of phenylephrine-precontrated Lmna +/+ and Lmna G609G/G609G aortic rings exposed to the endotheliumindependent vasodilator DEA-NO ( Figure 2B). These results thus indicate that endothelial dysfunction underlies impaired vessel relaxation in mice with ubiquitous progerin expression.
We next investigated which of the main factors contributing to acetylcholine-induced endothelial relaxation were altered in Lmna G609G/G609G aorta. For this, we prepared acetylcholine- (B) Strength of contraction induced by KCl. Statistical differences were analyzed by two-way ANOVA with Bonferroni's post-hoc test for phenylephrine data, and by two-tailed t-test for KCl data. * p < 0.05 *** p < 0.001.
To assess endothelial function, we first exposed phenylephrine-precontrated aortas to the endothelium-dependent vasodilator acetylcholine ( Figure 2A). Relaxation induced by the physiological acetylcholine dose (0.1 µM) was significantly lower in Lmna G609G/G609G vessel segments, a result also evidenced by a difference in logEC50 (−7.025 ± 0.06 in Lmna +/+ aortic rings versus −6.511 ± 0.09 in Lmna G609G/G609G aortic rings) (Figure 2A, left) and a lower AUC for acetylcholine-induced relaxation (Figure 2A, right). In contrast, there were no significant differences in the relaxation of phenylephrine-precontrated Lmna +/+ and Lmna G609G/G609G aortic rings exposed to the endothelium-independent vasodilator DEA-NO ( Figure 2B). These results thus indicate that endothelial dysfunction underlies impaired vessel relaxation in mice with ubiquitous progerin expression.
We next investigated which of the main factors contributing to acetylcholine-induced endothelial relaxation were altered in Lmna G609G/G609G aorta. For this, we prepared acetylcholine-induced relaxation curves in the presence of the following agents: L-NAME (NOS inhibitor), tranylcypromine (PGI 2 synthase inhibitor), catalase (H 2 O 2 decomposing agent), and Tempol (O 2 − scavenger). Significant inhibition of acetylcholine-induced relaxation was observed only in L-NAME-treated Lmna +/+ and Lmna G609G/G609G aortic rings; the other drugs had no significant effect irrespective of mouse genotype ( Figure 2C). These findings are consistent with the notion that NO is the main factor underlying Cells 2020, 9, 656 5 of 11 endothelium-dependent acetylcholine-induced relaxation in vessels of both genotypes and suggest that NO deficiency may account for the endothelial dysfunction in vessels of mice with ubiquitous progerin expression.
Cells 2020, 9, x 5 of 12 induced relaxation curves in the presence of the following agents: L-NAME (NOS inhibitor), tranylcypromine (PGI2 synthase inhibitor), catalase (H2O2 decomposing agent), and Tempol (O2 − scavenger). Significant inhibition of acetylcholine-induced relaxation was observed only in L-NAMEtreated Lmna +/+ and Lmna G609G/G609G aortic rings; the other drugs had no significant effect irrespective of mouse genotype ( Figure 2C). These findings are consistent with the notion that NO is the main factor underlying endothelium-dependent acetylcholine-induced relaxation in vessels of both genotypes and suggest that NO deficiency may account for the endothelial dysfunction in vessels of mice with ubiquitous progerin expression.

VSMC-Specific Progerin Expression Provokes Contractile Impairment, but Neither VSMC-Specific nor EC-Specific Expression Is Sufficient to Cause Endothelial Dysfunction
VSMCs and ECs are key cellular components of the vessel wall that play major roles in the regulation of vascular tone. To determine the relative contribution of these cell types to impaired vascular tone regulation in progeroid mice, we performed wire myography experiments in aortic rings isolated from Lmna LCS/LCS SM22αCre tg/+ and Lmna LCS/LCS Tie2Cre tg/+ mice, which express progerin predominantly in VSMCs and ECs, respectively [25]. Controls for both models were Lmna LCS/LCS littermates that do not express progerin [23]. Contraction in response to phenylephrine or KCl was significantly lower in aortic rings from mice with VSMC-specific progerin expression ( Figure 3A

VSMC-Specific Progerin Expression Provokes Contractile Impairment, but Neither VSMC-Specific nor EC-Specific Expression Is Sufficient to Cause Endothelial Dysfunction
VSMCs and ECs are key cellular components of the vessel wall that play major roles in the regulation of vascular tone. To determine the relative contribution of these cell types to impaired vascular tone regulation in progeroid mice, we performed wire myography experiments in aortic rings isolated from Lmna LCS/LCS SM22αCre tg/+ and Lmna LCS/LCS Tie2Cre tg/+ mice, which express progerin predominantly in VSMCs and ECs, respectively [25]. Controls for both models were Lmna LCS/LCS littermates that do not express progerin [23]. Contraction in response to phenylephrine or KCl was significantly lower in aortic rings from mice with VSMC-specific progerin expression ( Figure 3A), like in aortas from Lmna G609G/G609G mice with ubiquous progerin expression (cf. Figure 1A). In contrast, aortas expressing progerin only in ECs contracted normally when incubated with phenylephrine or KCl ( Figure 3C). Likewise, vasodilation induced by either acetylcholine or DEA-NO was not significantly different from controls in Lmna LCS/LCS SM22αCre tg/+ ( Figure 3B) or Lmna LCS/LCS Tie2Cre tg/+ aortic rings ( Figure 3D). These results demonstrate that smooth muscle, not endothelium, is the main vascular cell type driving contractile impairment in progeroid mice and suggest that progeroid endothelial dysfunction requires simultaneous expression of progerin in VSMCs and ECs. like in aortas from Lmna G609G/G609G mice with ubiquous progerin expression (cf. Figure 1A). In contrast, aortas expressing progerin only in ECs contracted normally when incubated with phenylephrine or KCl ( Figure 3C). Likewise, vasodilation induced by either acetylcholine or DEA-NO was not significantly different from controls in Lmna LCS/LCS SM22αCre tg/+ ( Figure 3B) or Lmna LCS/LCS Tie2Cre tg/+ aortic rings ( Figure 3D). These results demonstrate that smooth muscle, not endothelium, is the main vascular cell type driving contractile impairment in progeroid mice and suggest that progeroid endothelial dysfunction requires simultaneous expression of progerin in VSMCs and ECs.  We next tested whether mechanical impediment by the stiff extracellular cell matrix might contribute to contractile impairment in progeroid mice. Since collagen deposition has been shown to cause aortic stiffness in progeroid Lmna G609G/G609G mice [25], we examined KCl-induced aortic contractions before and after collagen disruption with collagenase. Treatment with collagenase did not improve KCl-induced aortic contraction in Lmna G609G/G609G and Lmna LCS/LCS SM22α tg/+ mice ( Figure  4A  We next tested whether mechanical impediment by the stiff extracellular cell matrix might contribute to contractile impairment in progeroid mice. Since collagen deposition has been shown to cause aortic stiffness in progeroid Lmna G609G/G609G mice [25], we examined KCl-induced aortic contractions before and after collagen disruption with collagenase. Treatment with collagenase did not improve KCl-induced aortic contraction in Lmna G609G/G609G and Lmna LCS/LCS SM22α tg/+ mice ( Figure 4A

Treatment with Sodium Nitrite Improves Vascular Function in Progeroid Mice
The key role of NO in progerin-induced endothelial dysfunction ( Figure 2C) suggested that NO supplementation might improve vascular tone regulation in progeroid mice. We therefore treated Lmna G609G/G609G mice and Lmna +/+ controls with drinking water supplemented with sodium nitrite (see Materials and Methods). Wire myography with isolated aortic rings showed that nitrite treatment restored sensitivity to acethylcholine-induced aortic relaxation in progeroid aortic rings ( Figure 5A, cf. Figure 2A). Nitrite treatment had no effect on DEA-NO-induced endothelium-independent Statistical differences were analyzed with two-way ANOVA followed by the Sidak multiple comparison test. * p < 0.05; *** p < 0.001.

Treatment with Sodium Nitrite Improves Vascular Function in Progeroid Mice
The key role of NO in progerin-induced endothelial dysfunction ( Figure 2C) suggested that NO supplementation might improve vascular tone regulation in progeroid mice. We therefore treated Lmna G609G/G609G mice and Lmna +/+ controls with drinking water supplemented with sodium nitrite (see Materials and Methods). Wire myography with isolated aortic rings showed that nitrite treatment restored sensitivity to acethylcholine-induced aortic relaxation in progeroid aortic rings ( Figure 5A, cf. Figure 2A). Nitrite treatment had no effect on DEA-NO-induced endothelium-independent relaxation in progerid or control mice ( Figure 5B, cf. Figure 2B). Nitrite treatment also ameliorated the impaired phenylephrine-dependent contraction in aortic rings from progeroid mice ( Figure 5C, cf. Figure 1A). In contrast, nitrite treatment did not normalize KCl-induced contraction, which remained significantly lower in progeroid aortic rings control aortic rings than in vessels from Lmna +/+ controls ( Figure 5B, cf. Figure 1B). These results demonstrate that dietary supplementation with sodium nitrite can ameliorate defective vascular tone regulation in progeroid mice.

Discussion
Aging and associated CVD constitute a major sanitary, societal, and economic challenge [1]. It is therefore of utmost importance to extend knowledge of the cellular and molecular mechanisms underlying vascular aging in order to design more effective diagnostic tools, prevention strategies, and therapies to promote healthier aging. The investigation of vascular aging in humans is challenging due to the difficulty of organizing long follow-up longitudinal studies in large population cohorts, as well as inherent complexity of post-analysis due to the concurrence with aging of other confounding cardiovascular risk factors, such as hypercholesterolemia, diabetes, hypertension, and obesity [2][3][4]. These limitations can be circumvented by studying premature aging syndromes characterized by accelerated CVD but lacking other confounding risk factors [5,6].
The HGPS mouse models generated over the last decade are excellent tools for studying cardiovascular aging over a relatively short time frame and without the presence of confounding cofactors, enabling the identification of the cell types most susceptible to progerin-induced CVD

Discussion
Aging and associated CVD constitute a major sanitary, societal, and economic challenge [1]. It is therefore of utmost importance to extend knowledge of the cellular and molecular mechanisms underlying vascular aging in order to design more effective diagnostic tools, prevention strategies, and therapies to promote healthier aging. The investigation of vascular aging in humans is challenging due to the difficulty of organizing long follow-up longitudinal studies in large population cohorts, as well as inherent complexity of post-analysis due to the concurrence with aging of other confounding cardiovascular risk factors, such as hypercholesterolemia, diabetes, hypertension, and obesity [2][3][4]. These limitations can be circumvented by studying premature aging syndromes characterized by accelerated CVD but lacking other confounding risk factors [5,6].
The HGPS mouse models generated over the last decade are excellent tools for studying cardiovascular aging over a relatively short time frame and without the presence of confounding cofactors, enabling the identification of the cell types most susceptible to progerin-induced CVD [6,22]. In the present study, we investigated vascular tone regulation in three HGPS models. We found severe contractile impairment in Lmna G609G mice with ubiquitous progerin expression, which was also observed in Lmna LCS/LCS SM22αCre tg/+ with VSMC-specific progerin expression, but not in Lmna LCS/LCS Tie2Cre tg/+ mice with EC-specific progerin expression. These findings identify VSMCs as the main cell type targeted by progerin to impair vessel contraction, consistent with our recent studies showing that VSMC-specific progerin expression is sufficient to fully recapitulate vascular alterations observed in mice with ubiquitous progerin expression, including vessel stiffness with inward remodeling; VSMC loss; increased collagen deposition and decreased transverse waving of elastin layers in the medial layer [25]; and accelerated atherosclerosis, medial LDL retention, and plaque vulnerability (when examined in a proatherogenic Apoe −/− background) [26,27].
The defective vasoconstriction in progeroid mice reported here is in agreement with results by others showing decreased vasoconstrictor responses in normally aged mice [40,41]. We recently identified collagen deposition by VSMCs as a major contributor to vessel stiffness in Lmna G609G/G609G mice [25]. We therefore hypothesized that progerin-dependent contractile impairment could be due to mechanical impediment by a collagen stiff matrix, smooth muscle cell degeneration, or be caused by a combination of both. Treatment of aortic rings with collagenase to disrupt collagen did not improve contractile responses in Lmna G609G/G609G and Lmna LCS/LCS SM22αCre tg/+ aortas, suggesting that collagen deposition is not the cause of impaired vasoconstriction in these animals. VSMC degeneration and dysfunction is therefore the most likely reason for contractile impairment, which might be the initiating cause of progerin-induced collagen deposition and defective vasoconstriction in HGPS models.
Our results show that progeroid endothelial dysfunction requires simultaneous expression of progerin in VSMCs and ECs, since progerin expression in VSMCs or ECs alone does not impair acetylcholine-dependent vessel relaxation. Moreover, vessels from mice with EC-specific progerin expression do not recapitulate the stiffness and inward remodeling observed in mice with ubiquitous or VSMC-specific progerin expression [25]. These findings indicate that the endothelial dysfunction observed in mice with ubiquitous progerin expression must be the result of systemic factors, or that it requires the combination of a dysfunctional medial and endothelial layer. However, since we have used a chemical signal to induce NO-dependent relaxation, we cannot rule out that other mechanically driven signals relevant for endothelial-dependent relaxation might be affected by EC-specific progerin expresion. Interestingly, transgenic mice with EC-specific overexpression of human progerin exhibit interstitial myocardial and perivascular fibrosis without VSMC loss [42]. Excessive collagen production in these mice is primarily derived not from ECs directly but from EC-dependent induction of a profibrotic response in the surrounding tissue [42]. HGPS patients show no alterations to flow mediated dilation [7,11], an indirect measure of endothelial function; nevertheless, the severe atherosclerosis in these patients must be preceded or accompanied by endothelial dysfunction at some point. Indeed, endothelial dysfunction and vascular stiffness might be induced independently but in parallel in HGPS, synergistically promoting vessel dysfunction and atherosclerosis as the disease progresses.
Deficient NO bioavailability is known to cause endothelial dysfunction in various CVD settings and in physiological aging [29,30]. In addition, mice overexpressing human progerin exclusively in ECs have reduced eNOS expression and NO levels [42]. We recently reported that NO supplementation by adding sodium nitrite to drinking water prevents vascular stiffness in progeroid mice [25]. In the present study, we demonstrate that NO is the main driver of endothelial-dependent vasodilation in mice with ubiquitous progerin expression and that treatment with sodium nitrite reverts endothelial dysfunction Cells 2020, 9, 656 9 of 11 and partially ameliorates contractile impairment in progeroid mice. Nitrite supplementation increases NO bioavailability without the requirement of L-arginine and NOS [43]. The beneficial effect of sodium nitrite in phenylephrine-induced contractility we observed in progeroid aortic rings might be related to an improvement in VSMC and EC homeostasis rather than to reduced vessel stiffness, since treatment with collagenase to decrease vessel stiffness did not abrogate progerin-induced defects in contractility. Future studies should examine whether the beneficial effect of nitrites on the contraction of progeroid aortic rings can be also explained by local compensatory mechanisms, such as a shift in NO/prostaglandin/EDHF balance.
In summary, our work demonstrates for the first time the presence of vascular tone abnormalities such as a severe VSMC contractile impairment and endothelial dysfunction in a mouse model of premature aging caused by ubiquitous progerin expression. VSMCs are the main cell type involved in this contractile impairment, whereas neither VSMC-specific nor EC-specific progerin expression are sufficient to provoke endothelial dysfunction, which likely requires progerin expression in both ECs and VSMCs, and possibly also in other cell types. Our results also suggest dietary supplementation with nitrites as a novel therapy to treat CVD in HGPS patients.