Minoxidil treatment reduces aorta stiffening in diabetic mice.
Numerous studies for more than a decade have demonstrated that metabolic diseases such as diabetes and/or obesity are pathologies contributing to premature aging of tissues by disrupting cellular processes (e.g. autophagy, apoptosis, telomere, senescence)[6, 61–63] but also extracellular processes (proteolysis)[27, 64]. In order to evaluate its efficiency in limiting the premature aging of the aorta wall observed in db/db mice, we administrated minoxidil to diabetic mice for eight weeks in conditions comparable to those used to stimulate neosynthesis/protection of arterial EFs in young rats [45] and aged mice [43, 65].
Minoxidil-reduced systolic and pulse blood pressures (not mean arterial pressure (MAP)), as well as aortic pulse wave velocity or compliance and distensibility measured using the high ultrasound method, were indicative of arterial stiffness (Figs. 2A to 2C, respectively). This was associated with a decrease in expression of the smooth muscle cells (SMC) contraction markers α-SMA, SM-22α, MLCK (Myosin light-chain kinase), Calponin and MYH11 (smooth muscle myosin heavy chain) (Figs. 2D and 2E, and supplemental Fig. 1B). These data demonstrated the antihypertensive and anti-aortic stiffening efficacy of minoxidil in diabetic animals but did not affect glycemic parameters (Table 1). Figure 2C suggests that minoxidil administration might be sufficient to restore an almost normal aortic function (observed by the dashed red line obtained with C57Bl6 mice (n = 10)). Minoxidil also induced aorta wall remodeling, leading to decreased intima-media and adventitia thicknesses (Fig. 3A and Supplementary 1B). This is possibly due to a decrease in total wall collagen content, particularly type III and I collagen as we observed by red picrosirius staining (Fig. 3B). The increase of the mRNA expression of these collagens (Fig. 3C) together with the absence of increased total collagen (Fig. 3D) contents and crosslinks (Fig. 3E) suggest the degradation of neosynthesized collagens.
Minoxidil treatment promotes elastogenesis while reducing elastolysis, in diabetic mice. Elastin autofluorescence level (Figs. 4A) and elastin quantity (Figs. 4B and supplemental Fig. 2)are increased, suggesting an increase in elastic fiber content and/or a decreased elastic fiber degradation after minoxidil treatment, as shown by elastin autofluorescence (Fig. 4A) and Hart’s staining (Fig. 4C). Indeed, while the number of elastic lamellae remained unchanged by minoxidil treatment (Fig. 4D). Likewise, the number of elastic lamella ruptures (Fig. 4E) is decreased by the treatment reflecting a restructuring of lamellae. This reduced fragmentation of the elastic networks is confirmed by a significant decrease in plasmatic elastin-derived peptides (EDP) and desmosine levels (Fig. 4F). Regarding elastolysis, the activities of cathepsin S and NE were reduced by the treatment (Fig. 4G), while the tissular proteinase mRNA levels were unaffected by minoxidil (Fig. 4H). The minoxidil-induced decrease in elastolysis in the aorta was amplified by the stimulatory effect of the treatment on the increased tissular expression of natural proteinase inhibitor, such as Serine peptidase inhibitor (SERPIN) (Fig. 4I). The fact that minoxidil treatment stimulated aortic elastogenesis is further suggested by elevated mRNAs levels for elastin, LTBP4, and LOXL1 (Fig. 4J). Increased and efficient elastogenesis was demonstrated by the increase of cross-links following the treatment (Fig. 4K), as the occurrence of these cross-links is an indicator of the maturity and functionality of EFs. Inhibition of elastolysis and induction of elastogenesis by minoxidil improved elastic fiber content and function, as indicated by the decrease of elastic lamellae and interlamellar space stiffnesses in diabetic mice (Fig. 4L). Taken together, these observations show that minoxidil limits premature aging of the aortic wall in diabetic mice and partially restores aorta function. Nevertheless, the cardiac parameter data (Fig. 2F) show that, following the administration of minoxidil, cardiac stroke volume and left ventricle end-diastolic volume increased in db/db mice. The data highlights the fact that problems associated with alterations in left ventricular (LV) function can eventually impact aortic function and structure over time. This could compromise the anti-vascular aging effects of the medications used in our study and others [43, 44, 66].
Chronic nebivolol treatment limits the progression of aortic stiffness induced by diabetes. Minoxidil chronic treatment can compensate elastolysis induced by diabetes. However, this treatment has several side effects on cardiac functions, which limit its use (see Fig. 2F) [67]. For this reason, we sought to test other antihypertensive molecules that could have similar beneficial effects without these harmful side effects. Among antihypertensive molecules, pharmacological blockade of the β1-adrenergic receptor can extend the life span of mice and flies, independently of body weight or metabolic syndrome [68]. Therefore, we chose to evaluate the effect of nebivolol, which has been described as a β-blocker reducing heart rate (Fig. 5A), and a potent vasodilator activating β2 adrenergic receptors in endothelial cells and SMCs [32, 69]. Figure 5B shows that treatment with nebivolol for eight weeks is sufficient to reduce systolic, mean, and pulse arterial pressures. We also observe nebivolol-induced decreases in aortic pulse wave velocity (Fig. 5C), which favors the compliance of thoracic aorta and decreases Young’s modulus (Fig. 5D), and expression of vasoconstriction factors such as SM-22α, α-SMA, MLCK, Calponin and Myh11 (Figs. 5E, and 5F). Interestingly, Figs. 5B to 5D demonstrate that nebivolol restores aortic functions, at levels observed in young, non-diabetic mice.
Nebivolol treatment protects EFs and stimulates neosynthesis in diabetic mice. From an anatomical point of view, treatment with nebivolol also significantly reduced adventitia and media-intima thicknesses (Fig. 6A and Supplementary 1B). Importantly, collagen modifications cannot explain by itself thicknesses reduction after treatment. Indeed, we noticed that the mRNA expression of types I and III were significantly increased (Fig. 2B) while collagen staining by red picrosirius, in the aortic adventitia was reduced (Fig. 2C). Moreover, total collagen level (Fig. 2D) and crosslinks quantities (Fig. 2E) were comparable to those observed in untreated db/db mice. Using Western blotting (supplemental Fig. 2), elastin autofluorescence (Figs. 7A) and extraction of insoluble elastin (Fig. 7B), we demonstrated an increase of elastin quantity after nebivolol treatment. Using Hart’s staining (Fig. 7C) or by elastin autofluorescence (Fig. 7A), we observed that, although the number of elastic lamellae was unchanged in the media (Fig. 7D), their integrity was improved by nebivolol treatment. This was evidenced by the dramatic decrease in elastic lamella ruptures (Fig. 7E), and the decreased plasma levels of EDP and desmosines (Fig. 7F). In parallel, nebivolol treatment decreased plasma cathepsin S and NE activities (Fig. 7G), while, surprisingly, the expression of cathepsin S within the aorta (Fig. 7H) was increased. The expression of the natural inhibitors of MMP9 and NE elastases, say TIMP1 and SERPIN, was significantly increased by nebivolol treatment (Fig. 7I). Nebivolol treatment also induced the expression of elastin and LOXL1 and decreased LOXL3 expression (Fig. 7J), suggesting a positive effect on elastogenesis. Figure 7K shows that this neosynthesis of elastin (Supplemental Fig. 2) was associated with an increase of all crosslink classes, suggesting the formation of mature and functional elastin. The consequence of the treatment was a spectacular drop in aortic rigidity (by 5–10 times), as measured by AFM (Fig. 7L). Altogether, these findings suggest that chronic treatment with nebivolol can limit the EFs premature aging, with fewer cardiovascular side effects.
Minoxidil and nebivolol treatments stimulate elastogenesis and limit elastolysis in cultured vascular SMCs. As described in the literature [70, 71], diabetes promotes in vivo vascular smooth muscle contraction processes and the expression of elastolysis markers. In contrast, the use of the antihypertensive molecules minoxidil or nebivolol in diabetic animals induced a decrease in the markers of SMC contraction and an increase in the markers of elastogenesis. In this context, we aimed to focus on the effects of the treatments, Minoxidil and Nebivolol, on the behavior of insulin-resistant smooth muscle cells (SMCs). For this purpose, SMCs were pre-incubated with a medium enriched in glucose-palmitate. To maintain consistency between in vivo models (male db/db mice from the C57/Bl6J strain) and in vitro conditions, we opted to utilize the MOVAS cell line, derived from SMCs of male C57Bl6J mice. Similar to endothelial cells, SMCs play a crucial role in preserving the homeostasis of the ECM. In Fig. 8, insulin was used as a positive control having the ability to induce hyperpolarization of SMC membranes [72, 73]. Glucose-palmitate treatment decreased the phosphorylation levels of the insulin receptor and related pathway actors, Akt and transcription factor FOXO1 (Fig. 8A). Palmitate-glucose condition also decreases glucose up-take and increases mRNA PEPCK expression, known to be under the control of FOXO1 activity (Supplemental Fig. 3). Together, these data suggested that palmitate-glucose condition induced an insulin-resistance in MOVAS cells. Surprisingly, insulin alone reduced the expression of α-SMA and increased those of SM-22α and h-Caldesmon, all markers of contraction (Fig. 8B). Insulin resistance induced by glucose-palmitate, alone or associated with insulin, minoxidil, or nebivolol, did not significantly influence the expression of SM22α or h-Caldesmon, except for nebivolol, which restored a level of h-Caldesmon expression close to that observed with insulin alone (in the absence of insulinoresistance). On the other hand, the glucose-palmitate condition significantly increased α-SMA expression whereas, when associated with insulin, minoxidil, or nebivolol, we observed a decrease in its expression, returning close to that of insulin alone (in the absence of insulinoresistance). However, glucose-palmitate did not induce insulinoresistance regarding α-SMA expression since insulin induces the same α-SMA mRNA level regardless of the presence or absence of glucose-palmitate. This suggests that in vitro insulinoresistance could favor the contraction of MOVAS cell line, isolated from the smooth muscle of an adult mouse, by changing their α-SMA levels. Figure 8C and Fig. 9A show that insulin stimulates mRNA expression of elastogenesis markers (elastin, fibrillin 1, fibulin 5, LTBP4, and LOXL1) and elastin production (Figs. 8D and 8E). Conversely, the induction of insulin resistance (despite the presence of insulin) does not elevate the expressions of these same markers or even decrease their expressions, with the possible exception of LTBP4. The addition of minoxidil or nebivolol in the presence of glucose-palmitate increases mRNA (Fig. 8C and Fig. 9A) and protein (Fig. 8F and 8G) levels of all elastogenesis markers except for LOXL1. Insulin alone has no effect on elastolysis markers, MMP-9 and cathepsin S (Fig. 9B). Glucose-palmitate-induced insulin resistance significantly increased the expression of both elastases, and this effect was abolished by minoxidil or nebivolol. Surprisingly, minoxidil and nebivolol also reduced the expression of MMP-9 natural inhibitor, TIMP1 (Fig. 9B). When we ratioed the expression of an elastase to that of its natural inhibitor (i.e., MMP9/TIMP1 and cathepsin S/cystatin C) (Fig. 9C), we observed that insulin resistance increased the cathepsin S/cystatin C ratio, and this effect was abolished by insulin, minoxidil, and nebivolol. Conversely, glucose-palmitate reduced the MMP9/TIMP1 ratio, and neither insulin, minoxidil, nor nebivolol could modify this effect (Fig. 9C). These data suggest that insulin resistance promotes MOVAS cell contraction and favors elastolysis, whereas smooth muscle relaxants, such as insulin, minoxidil, and nebivolol, are pro-elastogenic factors. In correlation studies conducted in db/db mice treated with minoxidil and nebivolol, a negative correlation was observed between contraction markers (αSMA and SM22) and elastogenic factors (elastin and LOXL1) (Supplemental Table 2).
Minoxidil and Nebivolol treatments open potassium channels in cultured vascular SMCs. Minoxidil was described as a potassium channel opener that hyperpolarizes cell membranes, causing vascular muscle relaxation and a consequent increase in blood flow [45, 74]. The vasorelaxant effect of nebivolol has been primarily attributed to endothelial-dependent mechanisms, including beta-adrenergic receptors. However, nebivolol present additional vasorelaxant properties. Thus, the involvement of the ATP-sensitive potassium channels (KATP) of the SMCs would be a second mechanism involved in the vasorelaxant response to nebivolol [45, 48, 75–78]. Nebivolol might appear to act indirectly on the channel by decreasing cytoplasmic ATP concentrations by inhibiting mitochondrial ATP synthase and/or increase of extracellular efflux of ATP (supplemental Fig. 4 and [38–41]). The opening of potassium channels by minoxidil or by nebivolol can be associated with the induction of elastogenesis and the inhibition of elastolysis. In contrast, supplemental Fig. 5 shows that the closure of the voltage-gated potassium channels by tetraethylammonium (TEA) or of KATP channel by glibenclamide [42] [43] is associated with a decrease in most markers (protein or transcripts) of elastogenesis while those of elastolysis are expressed. To determinate if contractile status of MOVAS cells is determinant factor, we induced membrane depolarization of cells by addition of KCl in culture medium. Prior to the study, we evaluated the cytotoxic effect of extracellular KCl addition on MOVAS cell survival (supplemental Fig. 6). Excess of extracellular potassium effectively induces cell contraction but does not seem to have any significative effect on elastogenesis and elastolysis (supplemental Fig. 5). On other hand, the presence of KCl (or glibenclamide) inhibits the effects of minoxidil or nebivolol on the expressions of transcripts or proteins such as elastin. Taken together, these results suggest that the configuration of the potassium channels (opened or closed) is a major element in the control of the elastogenesis-elastolysis balance. Therefore, to determinate the signaling pathway linking potassium channel and transcript expressions is important. Several studies [34, 79] suggested that the decrease of KATP channel function leads to FOXO-1 repression.
Minoxidil and Nebivolol treatments inhibit FOXO transcription factor in cultured vascular SMCs. We hypothesized that the KATP channel pathway could activate the transcription factor FOXO1, involved in premature aging. Interestingly, data from the literature [80, 81] suggest that insulin signaling pathways modulate FOXO1 activity, in accordance with our findings regarding the insulin signaling pathway (Fig. 8A). In addition, we have shown that variations in elastin expression follow variations in expression and phosphorylation of FOXO1 (Figs. 8F and 8G), depending on whether the SMCs are insulin-resistant or not, or treated or not with minoxidil or nebivolol. Thus, we initially undertook a transcriptomic study of FOXO1 and FOXO3 on MOVAS cells incubated in different media, stimulating the opening or closing of ATP-sensitive potassium channels (see Fig. 10A). In cells incubated in a classical medium (DMEM), the presence of the KATP channel openers minoxidil and nebivolol drastically reduced the expressions of FOXO1 and, for nebivolol only, FOXO3. Conversely, three KATP channels closing conditions (culture medium with glibenclamide, KCl, or glucose-palmitate) significantly increased the expression of FOXO1, whereas only KCl and glucose-palmitate elevated FOXO3 mRNA levels. KCl- or glucose-palmitate-supplemented culture medium, minoxidil, or nebivolol caused a significant decrease in FOXO1 and FOXO3 expressions compared to glucose-palmitate or KCl alone, while they decreased FOXO1 and increased FOXO3 expressions in the presence of glibenclamide compared to glucose-palmitate or KCl alone (Fig. 10A). Interestingly, FOXO1 has been described as a potential regulator of elastin [16] and MMP-9 expressions [82, 83]. Therefore, to confirm the impact of FOXO1 on ELN and MMP-9 expression, we used the FOXO1 transcription inhibitor AS1842856 (Fig. 10B). Inhibition of FOXO1 activity by AS1842856 in insulin resistance conditions (palmitate + glucose) increased elastin and decreased MMP-9 expression compared to palmitate and glucose alone. We observed similar effects of AS1842856 on elastin and MMP-9 expressions in a glibenclamide (or KCl)-supplemented culture medium compared to glibenclamide or KCl alone. Surprisingly, in conditions where KATP channels were opened by minoxidil or in the presence of nebivolol, the FOXO1 inhibitor AS1842856 reduced the expression of MMP-9 and elastin (Fig. 10B).